Enterocin 7420 and Sage in Rabbit Diet and Their Effect on Meat Mineral Content and Physico-Chemical Properties

Abstract

Rabbit meat has outstanding nutritional characteristics—it is a lean meat with low fat, cholesterol and sodium content, with high-biological-value proteins, potassium, phosphorus, selenium, iron and vitamin B12 level. The dietary inclusion of natural bioactive compounds can improve the quality of rabbit meat. The present study evaluated the effect of enterocin 7420 (Ent 7420) and sage (Salvia officinalis) extract on the quality and mineral content of rabbit meat. A total of 96 Hyla rabbits (aged 35 days) were divided into E (Ent 7420; 50 µL/animal/d), S (sage extract; 10 µL/animal/d), E + S (Ent 7420 and sage in combination) and control (C) groups. Additives were administrated in drinking water for 21 days. A significant increase in meat iron (p < 0.01) content was noted; phosphorus and zinc levels were also elevated in experimental groups, compared with control data. Ent 7420 and sage treatment reduced the calcium and manganese (p < 0.01) contents. The physico-chemical traits of rabbit meat were not negatively influenced by treatment. Based on these results, diet supplementation, mostly with Ent 7420 but also in combination with sage, could enhance the quality of rabbit meat mineral, with a focus on its iron, phosphorus and zinc contents.

Full text

Citation: Pogány Simonová, M.;
Chrastinová, L’.; Lauková, A.
Enterocin 7420 and Sage in Rabbit
Diet and Their Effect on Meat
Mineral Content and
Physico-Chemical Properties.
Microorganisms 2022,10, 1094.
https://doi.org/10.3390/
microorganisms10061094
Academic Editor: David
Rodríguez-Lázaro
Received: 21 April 2022
Accepted: 23 May 2022
Published: 25 May 2022
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4.0/).
microorganisms
Article
Enterocin 7420 and Sage in Rabbit Diet and Their Effect on
Meat Mineral Content and Physico-Chemical Properties
Monika Pogány Simonová1, * , L’ubica Chrastinová2and Andrea Lauková1
1Centre of Biosciences of the Slovak Academy of Sciences, Institute of Animal Physiology, Šoltésovej 4-6,
04001 Košice, Slovakia; laukova@saske.sk
2Institute for Nutrition, National Agricultural and Food Centre, Hlohovecká2, 95141 Lužianky, Slovakia;
lubica.chrastinova@nppc.sk
*Correspondence: simonova@saske.sk
Abstract:
Rabbit meat has outstanding nutritional characteristics—it is a lean meat with low fat,
cholesterol and sodium content, with high-biological-value proteins, potassium, phosphorus, sele-
nium, iron and vitamin B12 level. The dietary inclusion of natural bioactive compounds can improve
the quality of rabbit meat. The present study evaluated the effect of enterocin 7420 (Ent 7420) and sage
(Salvia officinalis) extract on the quality and mineral content of rabbit meat. A total of 96 Hyla rabbits
(aged 35 days) were divided into E (Ent 7420; 50
µ
L/animal/d), S (sage extract; 10
µ
L/animal/d),
E + S (Ent 7420 and sage in combination) and control (C) groups. Additives were administrated
in drinking water for 21 days. A significant increase in meat iron (p< 0.01) content was noted;
phosphorus and zinc levels were also elevated in experimental groups, compared with control data.
Ent 7420 and sage treatment reduced the calcium and manganese (p< 0.01) contents. The physico-
chemical traits of rabbit meat were not negatively influenced by treatment. Based on these results,
diet supplementation, mostly with Ent 7420 but also in combination with sage, could enhance the
quality of rabbit meat mineral, with a focus on its iron, phosphorus and zinc contents.
Keywords: enterocin; sage extract; feed additives; mineral profile; rabbit meat
1. Introduction
Rabbit meat is an excellent source of minerals and trace elements, such as potassium,
calcium, phosphorus, and selenium and has the highest concentration of iron in any type
of meat. It is rich in vitamins, mainly vitamin B3, B6, B12 and E, and in Omega-3 and six
fatty acids. Another advantage of rabbit meat is its low sodium level. For this reason, it is
recommended for children, pregnant women and people with high blood pressure. It also
contains easily digestible proteins, with low amounts of cholesterol and fat. Even though
rabbit meat naturally offers a remarkable nutritious quality, the dietary fortification of
rabbits with bioactive compounds has been an increasing trend in recent years, and rabbit
meat is becoming a functional food with its superior nutritional properties [
1
]. Amongst
natural feed additives, bacteriocins come to the forefront, not only as commonly used starter
cultures and preservatives in food industry, but also in the agriculture sector and veterinary
medicine to improve the animals’ health and productivity [
2
–
4
]. Bacteriocins, antimicrobial
substances produced mostly by lactic acid bacteria (LAB; [
5
]), are usually used in animals
to enhance their health status and productivity, due to stabilized intestinal microbiota
and mucosal immunity. Mostly, colicins, microcins, lacticin, garvicin and lantibiotic nisin
are used in aquacultures, ruminants, poultry and swine production [
3
,
6
,
7
]. Enterocins
(bacteriocins produced mostly by enterococci) also have a great antimicrobial and immuno-
stimulatory potential but until recently, mostly enterocin-producing strains were applied to
piglets and poultry [
8
–
10
]. Only enterocin (Ent) A (produced by the Enterococcus faecium
EK13/CCM7419 strain) was
in vivo
tested in Japanese quails [
10
]. Rabbits are also a
significant part of animal food production, and mostly probiotics and herbal extracts are
Microorganisms 2022,10, 1094. https://doi.org/10.3390/microorganisms10061094 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022,10, 1094 2 of 9
studied as potential feed additives in their nutrition [
11
–
14
]. To extend the knowledge
regarding bacteriocin applications in rabbit farms, nisin, gallidermin, and enterocins 4231,
7420, EF55, A/P and M were supplemented to the rabbits’ diet alone or in combination
with phyto-additives [
15
–
24
]. Most of these studies present the bacteriocins/enterocins’
effect on growth performance, intestinal microbial composition and enzymatic activity, gut
morphology and the immune response of rabbits. The monitoring of changes in rabbit meat
properties due to bacteriocins/enterocins applications is further limited [
25
–
29
]. Plants
(whole plants, leaves, seeds as feedstuff) and their extracts (applied as additives) are often
used in rabbit nutrition due to their ability to stimulate appetite, digestion, immunity
and physiological processes, as well as their strong antimicrobial, anti-inflammatory and
antioxidant effects. Among herbal extracts and phyto-additives, fennel, thyme, rosemary,
sage, and oregano leaves, seeds and extracts are the most often supplemented to diets of
rabbit meat, and meat products are enriched with them. There is also a growing interest
in sage (Salvia officinalis) plants, seeds and extracts for use in animal feeding due to their
oil content, which is a source of polyunsaturated fatty acid (PUFA-linoleic and
α
-linolenic
acid). Dietary administration with sage and its extracts/by-products could increase the
PUFA content of animal products (eggs, meat [
14
]). Several previous studies demonstrated
that a combined application of enterocins and sage extract did not have a negative influence
on the characteristics of rabbit carcasses [
25
,
27
]. Moving forward from these results, the
objectives of this
in vivo
study were to determine the effects of non-commercial Ent 7420
and sage extract administration in drinking water, both separately and in combination, on
the physico-chemical parameters and mineral composition of rabbit meat.
2. Materials and Methods
2.1. Animals, Experiment Design and Diet
The experiment was performed in cooperation with our colleagues at the National
Agricultural and Food Centre (NAFC, Lužianky-Nitra, Slovakia). All applicable interna-
tional, national and/or institutional guidelines for animal care were followed appropriately,
and the experimental protocol was approved by the Institutional Ethic Committee, and the
State Veterinary and Food Administration of the Slovak Republic (permission code: SK CH
17016 and SK U 18016).
A total of 96 weaned Hyla breed male rabbits, aged 35 days (average live weight
767.2 g
±
17.5) were divided into four groups (n= 24), each consisting of 6 replicates
(1 replicate/4 rabbits/2 cages, 1 cage/2 animals). Rabbits were housed in standard cages
(61 cm
×
34 cm
×
33 cm) in a closed building equipped with heating and a forced ventilation
system, which allowed the environmental temperature to be adjusted within the range of
20
±
4
◦
C and to a relative humidity (70
±
5%). The photoperiod was 16L:8D. The animals
were fed with a commercial pelleted basal diet for growing rabbits (Table 1) with access to
feed and water ad libitum during the experiment.
The rabbits in group E were administered enterocin Ent 7420 (a dose of 50
µ
L/animal/day,
with activity of 25,600 AU/mL, from day 0/1 to day 21) in their drinking water, through nip-
ple drinkers. The semi-purified Ent 7420 was prepared according to Simonováand Lauková[
29
],
and its activity was tested using the agar spot test according to De Vuyst et al. [
31
] against
the principal indicator strain E. avium EA5 (isolated from piglet in our laboratory) and
expressed in arbitrary units per mL (AU/mL). The rabbits in group S received sage plant
extract (Salvia officinalis extract containing of 24% thujone, 18% borneol, 15% cineole; Calen-
dula, NováL’ubovˇna, Slovakia) in their drinking water at a dose of 10
µ
L/animal/day. The
animals in the E + S groups were administered the combination of Ent 7420 and sage extract.
Based on our previous experiments, showing that these additives could be dissolved in
distilled water [
31
], the additives were first applied 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 C) had the same conditions, but without additives being applied to their
drinking water, and they were fed a commercial diet. The experiment lasted for 42 days.

Microorganisms 2022,10, 1094 3 of 9
2.2. Slaughtering, Physico-Chemical and Mineral Analysis
At days 21 and 42, 6 rabbits from each group (n= 6, 1 rabbit/1 replicate) were selected
based on daily weight measurement to ensure similar weight of animals (day 21; average
live weight: 1697.5 g
±
123.5; day 42; average live weight: 2595.2 g
±
169.8). Rabbits were
slaughtered after electro-stunning (50 Hz, 0.3 A/rabbit/4 s) in an experimental slaugh-
terhouse by cutting the carotid and jugular veins, and they quickly bled out. Longissimus
thoracis et lumborum (LTL) was separated by removing the skin, fat and connective tissue,
before being chilled and stored 24 h at 4
◦
C until physicochemical and mineral content
analysis started.
Table 1. Composition and ingredients of the basal diet.
Feed Ingredients (%) Chemical Composition, Minerals and Vitamins (g/kg Feed)
Dehydrated lucerne meal 36.0 Crude protein (N*6.25) 175.0
Extracted sunflower meal 5.5 Crude fiber 188.3
Oats 13.0 Fat 32.0
Wheat bran 9.0 Ash 66.40
Dry malting sprouts 15.0 Organic matter 847.5
Extracted rapeseed meal 5.5 Acid detergent fiber (ADF) 185.1
Barley 8.0 Neutral detergent fiber (NDF) 315.5
DDGS 5.0 Lignine 42.3
Sodium chloride 0.3 Hemicellulose 148.5
Premix minerals 11.7 Cellulose 148.8
Limestone 1.0 Starch 127.2
Calcium 7.5
Phosphorus 5.9
Metabolic energy (MJ/kg) 10.3
Abbreviations: DDGS, dried distilled grains with solubles.
1
Premix contains per kg: Calcium 6.73 g; phosphorous
4.13 g; magnesium 1.90 g; sodium 1.36 g; potassium 11.21 g; iron 0.36 g; zinc 0.13 g; copper 0.03 g; and selenium
0.2 mg. Vitamin mixture provided per kg of diet: Vitamin A 1,500,000 IU; Vitamin D3 125,000 IU;
Vitamin E
5000 mg; Vitamin B1 100 mg; Vitamin B2 500 mg; Vitamin B6 200 mg; Vitamin B12 0.01 mg; Vitamin K3 0.5 mg;
biotin 10 mg; folic acid 25 mg; nicotinic acid 4000 mg; and choline chloride 100,000 mg. The metabolizable energy
content was calculated using the equation of Wiseman et al. [30].
The ultimate pH was determined at 24 h post mortem using a Radelkis OP-109 mea-
suring device (Jenway, Felsted, UK) with a combined electrode penetrating 3 mm into
samples. Color measurements were taken on the LTL 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*) with a
Lab Miniscan (HunterLab, Reston, VA, USA) according to the CIE Lab standards. Lightness
measurements at room temperature were also taken. Total water, protein and fat contents
were estimated using a FoodScaneTM-Meat Analyser (FOSS Analytical, Hilleored, Den-
mark) by an FT IR method (Fourier Transform infrared Spectroscopy); expressed in g/100.
From these values, the energy value was calculated (EC (kJ/100 g) = 16.75
×
Protein content
(g/100 g) + 37.68
×
Fat content (g/100 g)); Strmiska et al. [
32
]. Water-holding capacity
(WHC) was determined by compress method at constant pressure [
33
]. The analyzed
samples (0.3 g in weight) were placed on filter papers (Schleicher and Shuell No. 2040B,
Dassel, Germany) with previously weighed tweezers. Together with the papers, samples
were sandwiched between Plexiglas 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 the
aspirating spot and the pure filter paper. The ash content was determined by mineralization
of the samples at 550 ◦C according to STN 570185.
Macro and micro element analysis samples were ashed at 550
◦
C, and the ash was
dissolved in 10 mL of HCL (1:3). Minerals were determined by AAS iCE 3000 (Thermo
Fisher Scientific, Waltham, MA, USA). The phosphorus content was determined by the
molybdovanadate reagent on Camspec M501 (Spectronic Campes Ltd., Leeds, UK).

Microorganisms 2022,10, 1094 4 of 9
2.3. Statistical Analysis
Treatment effects on the meat parameters were analyzed using a two-way analysis
of variance (ANOVA), followed by a Bonferroni post hoc test for pair-wise comparisons,
where appropriate. Fixed effects for the model included period and treatment and the
interaction between them. Random terms included cage. The statistical model included the
effects of period and treatment and their interactions. Data are expressed as means and
standard deviations (SD). Mean values within the same row with different superscripts
indicate a significant difference p
≤
0.05. All statistical analyses were performed using
GraphPad Prism statistical software (GraphPad Prism version 6.0, GraphPad Software,
San Diego, CA, USA).
3. Results
The physico-chemical characteristics of the LTL are shown in Table 2. Only the time
effect was noted on the meat energy value. No negative effect of tested additives was noted
on the analyzed parameters. Reduced levels (although not significant) of pH24, WHC and
energy values were found in all experimental groups compared to control data, while water
content slightly increased.
Table 2.
The effect of Ent 7420 (E), sage extract (S) and their combinative (E + S) application on the
meat physico-chemical characteristics of rabbits Longissimus thoracis and lumborum (LTL; mean
±
SD).
Parameter Day of
Experiment E S E + S C Significance of Effects
Treatment Time Interaction
pH 24 h after killing 21 5.82 ±0.06 5.88 ±0.01 5.84 ±0.09 5.90 ±0.01 1.0000 0.9741 1.0000
42 5.66 ±0.06 5.67 ±0.08 5.67 ±0.09 5.73 ±0.02
Water content (g/100 g) 21 75.17 ±0.11 75.03 ±0.06 75.20 ±0.26 74.97 ±0.47 1.0000 0.9503 1.0000
42 75.30 ±0.10 75.63 ±0.35 75.33 ±0.29 75.47 ±0.38
Protein content
(g/100 g) 21 22.50 ±0.00 22.63 ±0.06 22.33 ±0.25 22.63 ±0.38 1.0000 0.9689 0.9999
42 22.27 ±0.06 21.97 ±0.15 22.60 ±0.30 22.40 ±0.37
Fat content (g/100 g) 21 1.33 ±0.12 1.33 ±0.06 1.47 ±0.21 1.40 ±0.17 1.0000 0.9854 1.0000
42 1.43 ±0.15 1.40 ±0.20 1.07 ±0.15 1.23 ±0.06
Ash content (g/100 g) 21 1.00 ±0.00 1.00 ±0.00 1.00 ±0.00 1.00 ±0.00 1.0000 1.0000 1.0000
42 1.00 ±0.00 1.00 ±0.00 1.00 ±0.00 1.00 ±0.00
L* (lightness) 21 50.06 ±3.30 47.63 ±2.39 50.48 ±2.39 49.97 ±5.46 0.9996 0.9985 0.9983
42 48.65 ±5.75 51.42 ±1.31 46.06 ±5.74 51.88 ±3.24
a* (redness) 21 0.72 ±0.60 2.82 ±1.57 3.63 ±0.35 1.18 ±0.97 0.4560 0.0809 0.0693
42 1.64 ±0.86 0.55 ±0.20 0.93 ±0.43 1.30 ±0.40
b* (yellowness) 21 7.39 ±2.23 7.80 ±1.70 6.86 ±1.14 7.25 ±0.91 0.4954 0.4411 0.9302
42 7.27 ±0.12 7.26 ±0.38 5.80 ±1.09 7.14 ±0.94
Water-holding
capacity (g/100 g) 21 36.44 ±0.74 36.91 ±1.81 37.27 ±1.91 34.47 ±4.21 0.4967 0.5436 0.2302
42 35.86 ±1.56 37.08 ±1.07 34.04 ±1.93 36.09 ±3.50
Energy value (kJ/100 g) 21 427.12 ±4.35 429.35 ±1.89 428.89 ±8.10 431.86 ±10.70 0.8339 0.0060 0.3941
42 426.97 ±4.86 420.69 ±10.06 418.74 ±6.28 420.00 ±7.14
Ent 7420 and sage treatment reduced calcium (day 21; E vs. E + S: p< 0.05; E, S
vs.
C: p< 0.01
; day 42; S vs. E, E + S, C: p< 0.001; E + S vs. S, C: p< 0.001) and manganese
(day 21; E + S vs. E, S, C: p< 0.01; E, S vs. C: p< 0.01; day 42; E, S, E + S vs.
C: p< 0.01)
contents and significantly elevated iron levels (day 21; E vs. S: p< 0.001; E vs. E + S, C: p< 0.01;
Table 3). While sage application mostly influenced the macro minerals, with the highest
phosphorus (S), magnesium and natrium (E + S) levels, Ent 7420 (E) application elevated
potassium levels, while iron and zinc and reduced the calcium and copper concentrations,
demonstrating the highest/lowest values.

Microorganisms 2022,10, 1094 5 of 9
Table 3.
The effect of Ent 7420 (E), sage extract (S) and their combinative (E + S) application on the
meat mineral content of rabbits Longissimus thoracis and lumborum (LTL; mean ±SD).
Parameter Day of
Experiment E S E + S C Significance of Effects
Treatment Time Interaction
Calcium (mg/100 g) 21 6.20 ±0.01 a8.20 ±0.04 ab 11.30 ±0.04 bc 12.80 ±0.01 c<0.0001 <0.0001 <0.0001
42 8.13 ±0.01 a9.10 ±0.01 b7.73 ±0.01 ac 16.77 ±0.04 d
Phosphorus (mg/100 g) 21 201.93 ±0.44 228.47 ±0.12 218.77 ±0.06 206.20 ±0.18 0.0126 0.8122 0.3519
42 221.03 ±0.17 a185.63 ±0.26 b195.33 ±0.04 ab 225.33 ±0.09 b
Magnesium (mg/100 g) 21 25.10 ±0.01 25.37 ±0.01 25.40 ±0.01 24.77 ±0.01 0.6447 0.1187 <0.0001
42 26.73 ±0.01 26.30 ±0.01 27.07 ±0.01 26.13 ±0.01
Natrium (mg/100 g) 21 31.63 ±0.02 30.53 ±0.02 34.10 ±0.03 29.27 ±0.02 1.0000 1.0000 0.9684
42 29.07 ±0.02 29.73 ±0.01 25.17 ±0.01 29.50 ±0.01
Potassium (mg/100 g) 21 413.67 ±0.14 400.47 ±0.03 396.67 ±0.06 401.77 ±0.22 0.6994 0.3619 0.1928
42 411.53 ±0.16 410.73 ±0.10 407.43 ±0.03 406.30 ±0.11
Iron (mg/100 g) 21 0.579 ±0.035 a0.341 ±0.124 b0.465 ±0.092 b0.365 ±0.146 b0.0491 0.0088 0.2693
42 0.481 ±0.035 0.410 ±0.081 0.355 ±0.024 0.465 ±0.053
Manganese (mg/100 g) 21 0.064 ±0.004 a0.061 ±0.009 a0.066 ±0.025 b0.084 ±0.010 a0.0775 <0.0001 0.0524
42 0.029 ±0.012 a0.027 ±0.051 a0.018 ±0.007 a0.082 ±0.048 b
Zinc (mg/100 g) 21 1.350 ±0.244 1.123 ±0.141 1.216 ±0.143 1.043 ±0.190 0.2357 0.0126 0.0002
42 1.779 ±0.517 a1.647 ±0.504 a1.989 ±0.485 ab 1.190 ±0.131 b
Copper (mg/100 g) 21 0.117 ±0.017 a0.119 ±0.015 a0.195 ±0.025 b0.120 ±0.079 a0.0026 0.0519 0.0824
42 0.208 ±0.034 a0.109 ±0.001 b0.138 ±0.013 bc 0.200 ±0.066 ac
a,b,c–mean values marked with different letters differ significantly at p≤0.05.
4. Discussion
The Ent 7420 and sage application did not negatively influence the physico-chemical
properties of rabbit meat, similarly to previous results achieved through bioactive com-
pounds, such as the supplementation of bacteriocins, herbal extracts and beneficial strains
to rabbits [
26
–
28
,
34
–
36
]. Meineri et al. [
37
] and Rotolo et al. [
38
] also reported the adverse
effects of chia seeds and dried leaves on the traits of rabbit meat quality. However, no
significant changes within the tested parameters were determined. The pH and WHC de-
creased with increasing age, similar to findings presented by Pogány Simonováet al. [
34
,
35
]
and Koziol et al. [39]. The pH (acidity) of rabbit meat is an essential parameter, indicating
its shelf life and preventing the microbial growth (bacteriostatic effect of low pH), tech-
nological usability and quality of the rabbit meat, which also depends on many factors,
such as stress during transport and slaughter, the extent of debleeding and muscle type.
Although higher pH values of LTL samples were found when compared with our previous
studies, they were still under or at the upper limit of bibliographic values [
40
,
41
] and did
not negatively influence the positive quality of meat. There is a relation between pH and
WHC (increase in pH, increase in WHC); this finding was confirmed during the beneficial
E. faecium CCM7420 and EF9a strains administration to rabbits [
35
,
36
], while enterocins
and sage extract supplementation [
27
,
28
,
34
] showed the opposite effect (decreased pH, in-
creased WHC), as was also found in the recent experiment with Ent7420 and sage addition.
Lower energy values (but within the range of bibliographic values; [
1
,
40
]) of rabbit meat
were found compared to other enterocins and sage extract applications [
27
,
28
,
34
], but these
values were still higher than those after beneficial E. faecium strain supplementation [
35
,
36
].
Rabbit meat mineral content has a great variability. Most of tested minerals (except
calcium) were measured at lower levels than previously presented by Dalle Zotte and
Szendr˝o [
1
], Hermida et al. [
42
], and Nistor et al. [
43
]. Going forward from the increased
levels of most tested minerals in experimental groups, we hypothesize an enhanced nutrient
uptake from the intestine, and better mineral inclusion in rabbit meat. It was also interesting
to find out that the combined administration of Ent 7420 and sage affected some minerals,
such as phosphorus (S), iron, zinc (E), calcium and copper (E, S) in the opposite way when
compared to their separate applications. These findings suggest a more antagonistic effect
of tested compounds in the case of phosphorus (increased in E + S compared to lower levels
in E and decreased compared to higher level in S), iron and zinc (reduced in E + S compared
to elevated concentration in E and increased compared to lower S level). Regarding the
copper value, we assumed the synergistic effect of sage and Ent 7420 (higher level in
E+S
compared to E, S and C). Nevertheless, further experiments are needed to determine

Microorganisms 2022,10, 1094 6 of 9
and/or confirm in more detail if there is any other synergistic or antagonistic effect of
both additives on the tested minerals. It is known that probiotics and prebiotics can affect
intestinal mineral absorption by releasing bone-modulating factors such as phytoestrogens
from food [
44
]. This finding was also noted in this study regarding the bone minerals, but
mostly in the highest calcium level during the combination of Ent 7240 and sage extract in
rabbits which confirmed the supportive effect of Ent 7420 on higher phytoestrogens release
from sage enriched feed due to improved intestinal microbial environment. However, this
synergistic interaction of tested bioactive compounds on meat calcium level was noted in
E + S. The reduced Ca content compared to control Ca value did not confirm the positive
effects of phyto-estrogenic compounds in sage on Ca intestinal absorption (through estrogen
receptors within intestinal cells) and/or remodulation of serum Ca levels, as was previously
recorded by Pogány Simonováet al. [26]. Probiotics can increase mineral solubility via an
increased bacterial production of short-chain fatty acids (SCFA), enlarge the absorption
surface by promoting proliferation of enterocytes mediated by bacterial fermentation
products, improve gut health, and increase the expression of calcium-binding proteins,
mostly elevating calcium and magnesium absorption [
44
]. Another way to enhance mineral
absorption due to ionization and passive diffusion, is the acidic environment as a result
of a higher lactic acid formation [
45
]. Beneficial gut bacteria, also enhanced/optimized by
natural feed additives may improve the availability and absorption of polyvalent cations,
such as calcium, phosphorus, magnesium, zinc and iron due to optimum pH conditions
for enzymatic phytate degradation and reduction. This finding was repeatedly confirmed
after Enterococcus faecium CCM7420 and CCM8558 probiotic strains dietary inclusion in
rabbits, when phosphorus, iron and zinc concentrations in meat were increased compared
to untreated animals [
35
,
46
]. Bacteriocins in meaning postbiotics (metabolites of beneficial
bacteria) can balance/improve the host microbiome in favor of lactic acid bacteria because
of their antimicrobial activity, which inhibits the growth of enteropathogenic bacteria, and
thus ensures a higher lactic acid production. This may be another explanation of higher
iron and zinc intestinal absorption and the inclusion of rabbit meat, mostly as a result of the
higher iron level in meat samples from rabbits receiving Ent 7420. This hypothesis is also
confirmed by the results showing an increase in phosphorus, iron and zinc after enterocin
M application to rabbits [46].
In the case of iron content in food, it is important to know the respective amounts
of ferrous (heme; Fe
2+
) iron, sources from meat, liver and meat products, and ferric (non-
heme; Fe
3+
) iron, source from legumes, cereals, vegetables and fruits, with a dietary
transformation between these two states. Heme iron is the most bioavailable form of
iron, due to its coordination with a porphyrin ring hidden inside a globular protein, and
this arrangement protects iron from oxidation and insoluble precipitates forming in the
intestine, which promote its bioavailability [
47
]. Although there are only a few studies
concerning the probiotic and postbiotic/bacteriocin effect on rabbit meat, the achieved
results show an improvement of rabbit meat quality due to its higher iron content. Contrary
to us, Shah et al. [
48
] observed decreased iron and zinc content in rabbit hind leg samples
after microbial fermented feed utilization. On the other hand, these authors noted lower
copper and manganese levels, similar to our present and previous results [
27
,
35
,
46
]. Diet
supplementation with sage extract/chia seeds was shown to be effective in improving
rabbit meat nutritional quality, focusing on its fatty acid profile, and this meat can be
considered as a functional food [
1
]. Because there are only a few studies regarding the
effect of sage extract on rabbit meat minerals, we can only assume the activity of phenolic
compounds in increasing bone mineral content and bioavailability for iron [
49
]. Further
research will be necessary to clarify the effect of sage bioactive components on minerals
absorption and its inclusion in meat.
5. Conclusions
It seems that diet supplementation with Ent 7420 and sage extract was effective
in improving the meat mineral profile. While Ent 7420 significantly elevated the iron

Microorganisms 2022,10, 1094 7 of 9
content and increased zinc and potassium levels, sage extract beneficially influenced the
phosphorus and zinc concentrations of rabbit meat. Reduced calcium and manganese
levels were found after Ent 7420 and sage application. Both additives in combination also
increased phosphorus, iron, zinc and copper concentrations, without any adverse effects
on the physico-chemical properties of rabbit meat. We conclude that diet supplementation,
mainly with Ent 7420, can enhance the nutritional quality of rabbit meat.
Author Contributions:
Conceptualization, M.P.S. and A.L.; methodology, L’.C.; validation, M.P.S.;
investigation, M.P.S., L’.C. and A.L.; resources, M.P.S.; data curation, L’.C. and M.P.S.; writing—original
draft preparation, M.P.S.; writing—review and editing, M.P.S.; visualization, M.P.S.; supervision,
M.P.S. and A.L.; project administration, M.P.S.; funding acquisition, M.P.S. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by the Scientific Grant Agency of the Ministry for Education,
Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences VEGA, grant
number 2/0005/21.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Ethics Committee of the State Veterinary and Food
Administration of the Slovak Republic on 1 December 2016 (approval numbers SK CH 17016 and SK
U 18016).
Data Availability Statement: Data are available upon reasonable request to the corresponding author.
Acknowledgments:
We are grateful to P. Jerga for his skillful technical assistance and to J. Pecho for
slaughtering.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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