**1. Introduction**

In the last decade, the number of well-educated consumers focusing on a healthy lifestyle, diet and food sources is increasing. Rabbit meat is tender white meat, suitable for preparing delicious, nutritious, and mainly, healthy food, and greatly valued for its high protein level (20–21%) with essential amino acids of high digestibility, low fat content with a favorable proportion among saturated, monounsaturated, and polyunsaturated fatty acids, and it is almost free of cholesterol. Rabbit meat also provides a moderate amount of energy and low sodium content, but it is rich in potassium, magnesium, phosphorus, selenium, and B vitamins (as the richest source of vitamin B12 [1–3]). Due to these dietary properties, its frequent consumption is highly recommended, e.g., for pregnan<sup>t</sup> women, children, elderly people, and patients with cardiovascular disorders. Moreover, the fortification of the rabbit diet with functional compounds has an increasing tendency and, in this way, rabbit meat can represent a functional food [2]. The majority of studies have been focused on the qualitative parameters and sensory properties of rabbit meat [1,4–6]. Other works present the fatty acid profile, oxidative stability and lipid metabolism of rabbit meat, and their influence using natural additives [7–9]. Although the mineral content of rabbit meat has been investigated by several researchers [2,10–13], in vivo studies about the influence of natural feed additives on rabbit meat minerals are scarce [14,15].

In recent years, many alternatives—probiotics, prebiotics, enzymes, bacteriocins, organic acids, herbs, and their extracts have been tested in rabbits as feed additives to

**Citation:** Pogány Simonová, M.; Chrastinová, L'.; Lauková, A. Effect of *Enterococcus faecium* AL41 (CCM8558) and Its Enterocin M on the Physicochemical Properties and Mineral Content of Rabbit Meat. *Agriculture* **2021**, *11*, 1045. https:// doi.org/10.3390/agriculture11111045

Academic Editor: Wataru Mizunoya

Received: 9 September 2021 Accepted: 23 October 2021 Published: 25 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

enhance their productivity and health [2]. It is well known that probiotics—beneficial bacteria can improve gu<sup>t</sup> microbial balance, positively influence metabolism and nutrient digestibility as well as mucosal immunity, and maintain the health, growth, and productivity of animals. The majority of studies suggested a positive effect of dietary natural feed additives' inclusion on meat quality and that these compounds could be useful to improve nutritional properties—minerals, fatty, and amino acids of rabbit meat. While most of the works present the environmental, feeding, genetics, biological factors, and technological (pear-slaughter, transportation, processing) effects on rabbit carcass and meat quality [1], only a few of them have presented the effect of probiotics and/or bacteriocins on the rabbit meat mineral composition [14,15]; information about probiotic influence on meat of other monogastric animals, such as chickens and pigs is also rare [16,17]. Therefore, this study aimed to investigate the impact of feed administration by bacteriocin-producing and beneficial strain *Enterococcus faecium* AL41 (deponed to Czech Culture Collection of Microorganisms in Brno, Czech Republic, CCM8558) and its enterocin M (EntM) on the mineral content and quality of rabbit meat. Moreover, EntM is a new, not commercial bacteriocin, which can help to extend existing knowledge about bacteriocins' application in animal husbandry with a focus on the meat's nutritional quality—this is the novelty of this study.

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

#### *2.1. Experiment Schedule, Diet, Slaughtering, and Sampling*

The experiment was performed in co-operation with our colleagues at the National Agricultural and Food Centre (NAFC, Nitra, Slovakia). All applicable international, national and/or institutional guidelines for the care and use of animals were followed appropriately, and all experimental procedures were approved by the Slovak State Veterinary and Food Administration and Ethics Committees of both (permission code: SK CH 17016 and SK U 18016).

Seventy-two Hycole rabbits (weaned at age of 35 days, both sexes, equal male to female ratio per treatment) were used in this experiment. Rabbits were divided into 2 experimental groups (EG1, EG2) and 1 control group (CG), with 24 animals in each group. The average live weight of rabbits at the start of the experiment were 830.0 g ± 165.2 in EG1, 833.0 g ± 116.9 in EG2, and in CG it was 729.0 g ± 152.5. The rabbits were kept in standard cages (type D-KV-72, 61 × 34 × 33 cm, supplied by Kovobel company, Domažlice, Czech Republic) with two animals per cage. A cycle of 16 h of light and 8 h of dark was used through the experiment. Temperature (20 ± 4 ◦C) and humidity (70 ± 5%) were maintained throughout the experiment by heating and ventilation systems, and data were recorded continuously with a digital thermograph positioned at the same level as the cages. Animals were fed a commercial pelleted diet for growing rabbits (ANPRO-FEED, VKZII Buˇcany, Slovakia; Table 1) during the whole experiment with access to water ad libitum. Rabbits in group EG1 received bacteriocin-producing *E. faecium* CCM8558 strain possessing probiotic properties (1.0 × 10<sup>9</sup> CFU/mL) in their drinking water at a dose of 500 μL/animal/day for 21 days. It was marked by rifampicin to differ it from the total enterococci and prepared as described previously by Strompfová et al. [18]. The animals in group EG2 were administered EntM (prepared according to Mareková et al. [19]), a dose of 50 μL/animal/day, with activity 12,800 AU/mL for 21 days. Activity of EntM was tested by the agar spot test according to De Vuyst et al. [20] against the principal indicator strain *E. avium* EA5 (isolate from feces of piglet, our laboratory). The doses of additives and their manner of application were decided based on our previous in vitro studies testing the inhibitory activity of EntM against target bacteria and an experiment with rabbit-derived bacteriocin-producing strain *E. faecium* CCM7420 [21]. Based on our previous experiments, that these additives can be dissolved in distilled water and/or phosphate buffer [19], the additives were applied firstly to 100 mL of drinking water in all cages, and after consuming this volume, the rabbits had access to water ad libitum. Control rabbits (group CG) had the same conditions, but without additives being applied to their drinking water, and they were fed a commercial diet. Drinking water was provided through nipple drinkers. The experiment lasted for 42 days.

**Table 1.** Ingredients, chemical composition, and nutritive value of diets.


1 Premix provided per kg diet: vitamin A, 10,000 IU; vitamin D3, 2000 IU; vitamin E acetate, 30 mg; vitamin B2, 5 mg; vitamin B6, 2 mg; vitamin B12, 8 mg; Ca, 9.25 g; P, 6.2 g; Na, 1.6 g; Mg, 1.0 g; k, 10.8 g; Fe, 327.5 mg; Mn, 80 mg; Zn, 0.7 mg.

> At days 21 and 42, eight animals from each group were randomly selected for slaughter; they were stunned with electronarcosis (50 Hz, 0.3 A/rabbit/4 s) in an experimental slaughterhouse, immediately hung by the hind legs on the processing line, and quickly bled by cutting the jugular veins and the carotid arteries. After the bleeding, the *Longissimus thoracis* and *lumborum* (LTL) muscles were separated by removing the skin, fat and connective tissue, chilled, and stored 24 h at 4 ◦C until physicochemical analysis started.

#### *2.2. Physico-Chemical, Mineral, and Statistical Analysis*

The ultimate pH was determined 48 h postmortem (p.m.) with a Radelkis OP-109 (Jenway, Essex, UK) with a combined electrode penetrating 3 mm into the LTL. Color measurements were taken on MLD surface of the carcass at 24 h after bleeding. Color characteristics were expressed using the CIE L\*a\*b system (lightness-L\*, 0: black and 100: white), (redness and greenness-a\*; yellowness and blueness-b\*) using a Lab. Miniscan (HunterLab, Reston, VA, USA). Lightness measurements at room temperature were also taken. Total water, protein and fat contents were estimated using an INFRATEC 1265 spectroscope (FOSS, Tecator AB, Höganäs, Sweden) and expressed in g/100 g. The Near Infrared Transmission (NIT) principle is based on the fact that the measured sample absorbs the Near Infrared light at different wavelengths according to different characteristics such as fat or protein content [22]. From these values, the energy content was calculated [EC (kJ/100 g) = 16.75 × Protein content (g/100 g) + 37.68 × Fat content (g/100 g)]; [23]. Water holding capacity (WHC) was determined by compress method at constant pressure [24]. The analyzed samples (0.3 g in weight) were placed on filter papers (Schleicher and Shuell No. 2040B, Dassel, Germany) with tweezers previously weighed. Together with the papers, samples were sandwiched between Plexiglass plates and then subjected to a pressure of 5 kg for 5 min. The results were calculated from the difference in weight between the slips with aspirating spot and the pure filter paper. The ash content was determined by burning in Muffle furnace at 530 ± 20 ◦C according to STN 570185.

For macro and micro element analysis, samples were ashed at 550 ◦C, the ash was dissolved in 10 mL of HCl (1:3), and minerals were determined by AAS iCE 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Phosphorus content was determined by molybdovanadate reagen<sup>t</sup> on Camspec M501 (Spectronic Campes Ltd., Leeds, UK).

Treatment effects on tested parameters were analyzed using one-way analysis of variance (ANOVA) with Tukey post hoc test. All statistical analyses were performed using GraphPad Prism statistical software (GraphPad Prism version 6.0, GraphPad Software, San Diego, CA, USA). Differences between the mean values of the different dietary treatments were considered statistically significant at *p* < 0.05. Data are expressed as means and standard deviations of the mean (SD).
