**1. Introduction**

Bioactive peptides are genuine or generated components of ready-to-eat foods that may exert a regulatory activity in the human organism, regardless of their nutritive functions [1].

It is known that bovine milk is the most significant source of food-derived bioactive peptides [2]. The existence of bioactive peptides in fermented milk products and ripened cheese has been described [3]. During proteolysis, various peptides are released from the milk proteins; they are inactive while encrypted in the milk proteins. Proteolysis takes place during food processing, e.g., milk fermentation and cheese maturation, or during gastrointestinal transit. Some of the bioactive properties reported in peptides derived from milk products are antihypertensive, antioxidant, antimicrobial, immunomodulatory, and mineral binding [4–6]. The amount and type of bioactive peptides in cheese are a ffected by the starter culture and ripening conditions [7].

In 2019, the production of fluid milk in Mexico was approximately 12.6 million metric tons, of which almost 50% was utilized for cheese elaboration [7]. According to SAGARPA (Mexican Ministry of Agriculture and Sustainable Development) [8], in 2018, Mexican cheese production was 418,650 tons, where panela represented the third most-produced cheese and represented 11.7% of total production.

Panela cheese is a very popular handcrafted Mexican white, soft, fresh cheese manufactured from pasteurized cow's skim or partially skimmed milk [9], with little or no starter culture acidification [9,10]. According to the manufacture characteristics of this type of cheese, it is expected some degree of proteolysis and thus a release of bioactive peptides during manufacturing procedures, storage, and post-consumption.

On the other hand, hypertension is a state of a sustained increase in blood pressure (BP), related to cardiovascular diseases. Hypertension is the mortality most related factor around the world [11]. This is a chronic disease derived from many factors such as genetics, excessive sodium intake, age, smoking, sedentary lifestyle, and chronic diseases such as diabetes and obesity [12,13]. According to W.H.O. [14], more than one of every five adults su ffers from hypertension.

Within the organism, the regulation of blood pressure is related to the hormone "renin-angiotensin system" (RAS). The angiotensin-converting enzyme (ACE) is key within RAS because it converts the peptide angiotensin I to the vasoconstrictor angiotensin II, which tightens the blood vessel and increase the BP. ACE-inhibitors are competitive substrates for ACE, and among them are milk-derived bioactive peptides. The C-terminal of the inhibitor is the primary feature governing the inhibition of ACE [11].

Oxidative stress is a condition of imbalance between reactive oxygen species (ROS) with unpaired electrons and the body's ability to detoxify and repair the damage of the reactive components. It is widely related to the illnesses of the human body, including hypertension and other chronic diseases. Milk protein-derived peptides are among the natural dietary sources of antioxidants. Peptides from β-casein and <sup>α</sup>s1-casein are potent anion radical scavengers [11].

This work aimed to measure antioxidant, and ACE inhibition of a Panela cheese added (LSB-c)/not added (C-c) with probiotics. Physicochemical and textural parameters were also monitored during 15 days of storage (4 ± 0.6 ◦C) to evaluate their e ffect and relationship upon structural changes in both types of cheeses.

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

#### *2.1. Cheese Manufacture*

For this study, one 80-L batch of whole milk was obtained in the Tecnológico de Monterrey experimental agricultural field (CAETEC) (Querétaro, Mexico) and transported to the Tecnológico de Monterrey, Querétaro, Mexico under controlled temperature conditions. In the CAETEC, milk production is controlled to avoid composition variation throughout the year. To achieve this, the calf diet is standardized with a feed formulate by Tecnologico de Monterrey. The herd has about 100 milking cows. Di fferent national associations and industrial clients have certified the homogeneity and quality of CAETEC milk.

Whole milk (3.11% protein, 3.19% fat, initial pH 6.69) was cooled and stored at 4 ◦C for 24 h before cheese making. For cheese making, the Querataro's traditional panela making procedure was followed. Milk was pasteurized in a big pot (63 ◦C for 30 min) previously to cheese manufacturing and was split into two portions of 40-L at the food engineering facilities of Tecnológico de Monterrey, Querétaro, Mexico. The experimental design was a unifactorial design where the factor evaluated was storage time with four levels (0, 5, 10, and 15 days). Cheeses with probiotics (LSB-c) and without probiotics (C-c) where considered as independent blocks.

For the cheese added with probiotics (LSB-c), after pasteurization, the 40 L of milk was heated gradually to 32 ◦C, and 10<sup>9</sup> CFU/L of commercial type MM101 (Lyofast ®, Sacco, Via Manzoni, Italy) culture was added, which consisted of *Lactococcus lactis*, *Lactococcus cremoris*, *Lactococcus diacetylactis*, and *Streptococcus thermophilus*. Then, 10<sup>9</sup> CFU/mL *Bifidobacterium animalis* ssp lactis (Lyofast ®, Sacco, Via Manzoni, Italy) was added to the mix. Inoculated milk was kept at 32 ◦C for three h until it reached a pH value of 6.4. CaCl2 (Cal-Sol501, Industrias Cuamex, San Miguel Iztapalapa, CDMX, Mexico) was added by diluting 7 mL in 35 L of milk and kept in slow agitation for 1 min. Liquid calf rennet (Strength 1:7500. Qualact ®, Altecsa SA, Mexico City, Mexico) was added to milk (1.95 mL + 30 mL water). Coagulation of milk was completed in 30–35 min; then, the curd was cut into cubes (1 cm3) and allowed to rest in whey for 5 min before draining approximately 2/3 of whey. Salt was added to

curd at 1% and mixed manually for 5 min. Then cheeses were molded in 100 g plastic molds. Molds were slightly pressed for 20 min (by each side) by piling them up on one another, allowing natural whey drainage at room temperature. For C-c (control cheese, without any added culture), 40 L of milk were used following the above procedure without culture addition. Both kinds of cheeses were made simultaneously to avoid changes in milk quality. For each kind of cheese, 34 × 100 g-pieces were obtained, giving a total of 68 × 100 g pieces of cheese.

Individual cheeses were packed in plastic bags. Then, they were stored in refrigeration at 93.8 ± 1% of relative humidity (RH) and 4 ± 0.6 ◦C temperature for 15 days.
