**1. Introduction**

Enzymatic milk coagulation is a key step in cheese manufacturing and involves the addition of chymosin (rennet), an aspartate proteinase that is active in the stomach of non-weaned calves [1], and which hydrolyses the link between amino acids 105 (methionine) and 106 (phenylalanine) of the k-casein. Given various social (i.e., veganism) and religious (Islam, Judaism) issues, which entail limiting or reducing the use of chymosin, new sources of coagulants are needed.

Proteolytic enzymes extracted from plants may be an interesting alternative to animal rennet in dairy technology. In fact, milk-clotting enzymes have been identified in various plant species, such as *Lactuca sativa* [2], *Albizia lebbeck*, *Helianthus annuus* [3] and *Cynara cardunculus* [4].

Actinidin (EC 3.4.22.14) is a cysteine protease from kiwifruit (*Actinidia deliciosa*) with a wide pH activity range (4–10) [5]. Lo Piero et al. [5] demonstrated that actinidin forms milk clots with the typical conditions used in cheese manufacturing (optimum activity at 40–42 ◦C, mildly acidic pH values). The preferred substrate for actinidin is β-casein, followed by k-casein, and the result of this hydrolysis is the production of a small number of larger peptides [5]. Saha and Hayashi [6] revealed that dairy products that use kiwifruit juice actinidin have a lower perceived o ff-flavour.

Exploiting kiwifruit in milk cheesemaking could meet the goals of circular agriculture through the use of undersized and/or damaged kiwifruits in a simple and economically sustainable procedure

for the production of a clotting mixture. In addition, it could improve the nutritional/nutraceutical characteristics of the cheese, given that kiwifruit contains hydro-soluble components, such as vitamin C and polyphenols [7–9].

Several works have dealt with the clotting properties of kiwifruit. Katsaros et al. [10] reported that 1 mL juice rich in actinidin obtained from peeled, pulped and centrifuged kiwifruit corresponded to 0.42 ± 0.02 units, while 1 g of freeze-dried powder corresponded to 520 ± 20 units. Mazorra-Monzano et al. [11] compared kiwi, ginger and melon with chymosin and found that the kiwi extract produced the most similar curd in terms of texture to curd from chymosin. Lo Piero et al. [5] characterized the role of purified actinidin in casein proteolysis and reported that the activity for total casein was 129.6 U/mg and that the specific milk clotting activity (AC) is higher than the general one (AP); the AC/AP ratio (1.1) was similar to calf rennet (1.0) and much higher than microbiological rennet (0.22). Puglisi et al. [12] reported that kiwi juice shows twice the specific activity for total casein than purified actinidin of 299 U/mg, and speculated that, in kiwi, other proteases assist actinidin in the proteolysis process.

To the best of our knowledge, no data are available on the relationship between using kiwifruit extract in cheesemaking and the nutraceutical substances and flavour of cheese obtained with sheep and buffalo milk. We assessed the technological, nutraceutical and organoleptic properties of sheep and buffalo cheese made from kiwifruit extract compared with animal rennet.

#### **2. Materials and Methods**

#### *2.1. Experimental Design*

The study was carried out on 12 cheeses obtained from fresh and pasteurized milk, according to the following procedure: three buffalo milk cheeses were made with calf rennet (BM-C); three buffalo milk cheeses were made with kiwifruit extract (BM-K); three sheep milk cheeses were made with calf rennet (SM-C); and three sheep milk cheeses were made with kiwifruit extract (SM-K). The experiment was replicated twice in two independent batches. Milk was obtained from two neighbouring farms located in the province of Grosseto (southern Tuscany, Italy).

The kiwifruit extract was obtained by a modification of the method described by Katsaros et al. [10]. Briefly; 1 kg of kiwi pulp (*A. deliciosa*, cv. Hayward) were filtered through a cotton gauze. The filtrate was centrifuged (10,000 rpm for 15 min at 4 ◦C), and the supernatant was filtered using a 0.45 μm filter. The purified solution was lyophilized to obtain a powder rich in actinidin; the protein content of the powder was quantified by Bradford's method [13].

The calf rennet used for the coagulation of the BM-C and SM-C cheeses was NATUREN ® PLUS 215 (activity = 215 IMCU/mL; chymosin 63%; pepsin 37%) (CHR HANSEN, Hoersholm, Denmark).

Milk (200 mL) was clotted after the adjustment of the pH value by the addition of *Streptococcus thermophilus* CRV00 LYO 100 L starter (Santamaria srl, Burago di Molgara, Italy) (0.216 g per 100 mL) at 40 ◦C. Coagulants were added when the milk pH was 6.35–6.38. Calf rennet was diluted in distilled water to have a strength of 40 IMCU/mL. In order to have the same enzymatic activity (measured as Katsaros et al. [10]), for 200 mL of milk we used 80 mg of freeze-dried powder rich in actinidin.

After the milk had clotted, the curd was manually cut with a knife and stirred gently. Then, after about 10 min the cheeses were drained and put into moulds, which were turned and pressed manually. Finally, in order to eliminate the excess water, the miniature cheeses [11] were incubated at 40 ◦ C for 30 min. The cheeses were not salted. The weight of each cheese was recorded at the end of the cheesemaking and after 24 h. The syneresis of the cheeses was used as one of the indices of the level of coagulation, which was expressed as the volume of the whey (mL) per total mass (100 g) of the coagulated milk product. Cheese yields (initial and after 24 h) were calculated as curd weight (initial or after 24 h)/milk weight × 100.
