Abstract
This study aimed to examine the effects of oats cultivated in saline
and non-saline environments on the meat quality and muscle metabolism
of Qinghai Tibetan sheep. First, targeted and untargeted metabolomics
were used to examine oat quality and metabolites. Second, sheep muscle
quality and metabolites were analyzed. Finally, a combined examination
of the quality of the oats and their metabolites, as well as that of
the muscles, was compared with saline oats. This study hypothesizes
that, compared with non-saline environments, soil salinization can
improve the nutritional quality of oats, thereby enhancing the meat
quality and muscle metabolism of Qinghai Tibetan sheep. Saline-grown
oats were shown to have higher levels of crude protein, crude fat, free
amino acids, and simple sugar. The meat quality of the sheep fed on
saline oats was higher due to free amino acid and carbohydrate
metabolism, resulting in improved texture, color, water-holding
capacity, and cooked meat percentage, with lower steaming loss. The
findings of this study confirm the hypothesis that salinization
improves Tibetan sheep meat quality by optimizing oat composition,
providing a reference for agricultural and animal husbandry production
in saline areas.
Keywords: saline land, growing oats, Tibetan sheep, meat quality,
metabolomics
1. Introduction
The formation of muscle quality is regulated by feed nutrition. As the
core feed for ruminants, forage has metabolomic characteristics (such
as amino acids, fatty acids, and secondary metabolites) that enter the
animal body through digestion and absorption, directly participating in
material synthesis and energy metabolism in muscle cells and ultimately
affecting the flavor, tenderness, and nutritional composition of meat
[[40]1].
Saline soils represent a specialized habitat in terms of biological
resources and biodiversity [[41]2]. Saline–alkaline soils are typically
rich in soluble salts, such as Na^+, Cl^−, SO[4]^2−, and exchangeable
Na^+, with a total salt content significantly higher than that of
non-saline–alkaline soils. High concentrations of Na^+ can cause an
increase in pH, triggering an imbalance of mineral elements. This
further leads to elevated contents of mineral elements such as Na, Cl,
Ca, and Mg in saline–alkaline soils while reducing the availability of
trace elements such as Fe, Mn, and Zn [[42]3]. Changes in the
availability of mineral elements in the soil can affect the optimal
growth and functional status of plants. The saline–alkaline land in
Qinghai Province constitutes 3.2% of the national saline–alkaline land
area. The grass in these regions is suitable for the soil conditions,
exhibiting enhanced tolerance to cold, drought, and salinity, and
serves as feed for animals such as horses, cattle, and sheep [[43]4].
As the primary forage in the saline–alkaline land of Qinghai Province,
oats (Avena sativa L.) possess a high relative forage value (RFV). Oats
grown in saline–alkaline land are rich in essential nutrients,
including fats, proteins, and vitamins. They are renowned for their
tolerance to cold, drought, and saline–alkaline conditions and are
widely regarded as a viable option for the improvement of
saline–alkaline land [[44]5]. Liu et al. [[45]6] studied different oat
varieties grown in saline and alkaline soils of the Songnen Plain,
observing that these oats had high stem-to-leaf ratios, high RFV
values, and good relative feeding values. For this reason, they can
serve as pioneer plants for the improvement of saline–alkaline soils.
Bai et al. [[46]7] investigated the effects of alkaline stress on oats.
They found that under alkaline stimulation, the activities of
superoxide dismutase (SOD) and peroxidase (POD) in oats increased,
along with an increase in the content of soluble sugars. These
responses can enhance the alkaline tolerance of oats. Moreover, Yang et
al. [[47]8] found that drought stress (DS) can induce peroxidation and
osmotic stress in plants, which in turn respond to drought stress by
synthesizing osmoprotectants to regulate osmotic pressure. There is
insufficient understanding of the disparities in the quality of oats
grown on saline compared with non-saline soils in Qinghai.
Tibetan sheep (Ovis aries), one of China’s three major basic sheep
breeds, is an endemic species to the Qinghai–Tibet Plateau [[48]9].
These sheep are indigenous to Qinghai Province, where they are reared
by farmers and herders. They constitute a fundamental basis of the
animal husbandry industry in the province [[49]10]. Liang et al.
[[50]11] showed that feeding saline forage to sheep can improve the
growth performance and feed-to-weight ratio, serum protein metabolism,
immunity, antioxidant capacity, and mineral- and flavor-associated
compounds in the meat. Qiu et al. [[51]12] investigated the effect of
saline alfalfa (Medicago sativa L.) on the meat quality, organ
development, and serum biochemical indices of meat goats, finding that
saline alfalfa was more palatable than conventional alfalfa, with
better color, freshness, and tenderness of the meat, and reduced values
of cooking and dripping loss. A study by Moreno et al. [[52]13] showed
that the meat of lambs fed on saline oats had higher contents of ash,
total saturated fatty acids, and polyunsaturated fatty acids, together
with lower n-6:n-3 ratios. Pearce et al. [[53]14] discovered that the
carcasses of goats fed with halophytes, such as saltbush (Atriplex
spp.), can yield a higher proportion of lean meat and a lower amount of
fat. Furthermore, this method of feeding can increase vitamin E levels,
which helps to maintain the meat’s color. Therefore, it was thought
that the high metabolite content of oat grasses growing in salt water
could improve the quality of Tibetan lamb by encouraging the sheep to
store more nutrients and metabolites in their muscles.
There is currently no research on the impact of oat grasses cultivated
in saline–alkaline parts of Qinghai on the quality of Tibetan sheep
meat. Furthermore, there is no association between saline oats and meat
quality. To clarify the mechanism by which oat cultivation in saline
soil affects the quality of Tibetan sheep meat, and to identify the key
components in oats that influence this change, we conducted the
following experiments. Samples were obtained from both saline and
non-saline areas in Qinghai Province to investigate nutrient levels and
metabolites in oat plants. After a 120-day feeding period, the
longissimus dorsi muscles of Tibetan sheep were collected to analyze
their eating quality, nutritional quality, and targeted and untargeted
metabolites. The relationships between the differential metabolites of
the saline oats and the quality of Tibetan sheep, as well as among the
differential metabolites, were also examined. The principal metabolites
and metabolic pathways by which saline oats affect the quality of
Tibetan sheep were identified. The findings serve as a standard for
advancing the animal husbandry ecosystem in the Qinghai region,
offering extensive data and technical assistance for integrating
specific nutrients into standardized feeding protocols.
2. Materials and Methods
The Committee of Experimental Animal Care approved all experimental
procedures involving animals, while the Qinghai University of Animal
Care approved the handling techniques (QUA-2022-0515).
2.1. Samples Collection
2.1.1. Oat Sample Collection
Following the standards set by the National Forage Testing Association
(NFTA), oats were collected from the saline–alkaline land of Gonghe
County, Hainan Prefecture, Qinghai Province, China (GX; latitude:
36°28′2″ N; longitude: 99°16′26″ E; and altitude: 3168.1 m), and
non-saline land of Haiyan County, Haibei Prefecture, Qinghai Province,
China (YX; latitude: 36°59′36″ N; longitude: 100°55′5″ E; and altitude:
3111 m), and were harvested and preserved at −80 °C for subsequent
analysis of oat quality across the distinct regions. The soil sampling
areas were consistent with the oat-growing areas, and soil samples were
collected in the oat-growing fields of the two locations.
2.1.2. Experimental Design of Oat Feeding in Different Areas of Cultivation
Sixty healthy male sheep, 2 months old and with similar body
conditions, were selected and randomly allocated to the Gonghe-housed
group (YB, n = 30) and the Hai-yan-housed group (B0, n = 30). Group YB
sheep were raised at Xiangka Meiduo farm in Gonghe County, Hainan
Prefecture, Qinghai Province, China, and were fed local
saline-cultivated oats. Group B0 sheep were raised at Jinzang Farm in
Haiyan County, Haibei Prefecture, Qinghai Province, China, and were fed
local non-saline-cultivated oats. Referring to the literature with
minor modifications [[54]15,[55]16], both groups were provided with
identical feed concentrates and intake, as detailed in [56]Table 1. The
two groups of animals (30 in each group) were housed in enclosures with
wind-sheltered exercise areas that were also sunny, dry, and well
ventilated. The animals were fed twice daily at 08:30 am and 4:30 pm
with unrestricted access to feed and water; any feed remaining from the
previous feeding time was collected and weighed before the next
feeding. Furthermore, the housing was swept, gutters were cleaned
daily, and the housing and exercise yards underwent weekly disinfection
and sterilization. The fences were maintained in a clean and hygienic
condition. All Tibetan sheep underwent immunization, and the
transmission of internal and external parasites was systematically
prevented and managed. The official 120-day experiment was conducted
after a 7-day adaptation period, and slaughtering was initiated at 6
months of age.
Table 1.
Diet composition and nutritional level (dry matter basis, %).
Dietary Composition B0 (%) YB (%)
Corn 27.6 27.6
Soybean meal 3.6 3.6
Canola meal 6.6 6.6
Cottonseed meal 9.6 9.6
Wheat 7.8 7.8
Sodium chloride 0.6 0.6
Limestone 0.6 0.6
Sodium bicarbonate 0.6 0.6
Premix 3 3
Total 60 60
Roughage 40 (Haiyan oats) 40 (Gonghe oats)
[57]Open in a new tab
Note: YB represents feeding with oats grown in saline–alkali land, and
B0 represents feeding with oats grown in non-saline–alkali land. The
same below.
2.1.3. Tibetan Sheep Meat Sample Collection
At the end of the feeding trial, six experimental animals were randomly
selected from each group and transported to a nearby commercial
abattoir. The animals were fasted for 12 h (no food or liquid) and were
humanely slaughtered according to animal welfare procedures; i.e., the
lambs were stunned and bled. After slaughtering, the Longissimus dorsi
lumborum was also removed from one side of each carcass. Slaughtering
and sampling were performed together by professionals following uniform
standards. All samples were placed in dry ice and transferred to the
laboratory for storage at −80 °C for subsequent analysis. Six
replicates per group were used for all meat and metabolomics analyses.
2.2. Determination of Soil Mineral Elements
Following the method described by Song et al. [[58]17], the
concentrations of mineral elements in soil samples were determined
using inductively coupled plasma optical emission spectrometry
(ICP-OES, Optima 8300, Perkin Elmer, Waltham, MA, USA).
2.3. Oat Quality Analysis
2.3.1. Nutritional Values Analysis of Oats
The oats were dried in a blast-drying oven, pulverized, and passed
through a 40-mesh sieve to determine their value. Then, according to
the method described by Han et al. [[59]18], the moisture content was
determined using the oven method (DHG-9070A, Shanghai Bluepard
Instruments Co., Shanghai, China), the crude protein content was
determined using the Kjeldahl method (K9840, Hanon Advanced Technology
Group Co., Jinan, Shandong, China), and the crude fat content was
determined using the Soxhlet extraction method (SOx406, Shandong
Haineng Scientific Instrument Co., Jinan, Shandong, China).
Near-infrared (NIR) spectroscopy (INFRAMATIC 8620) was used to
determine acid detergent fiber (ADF) and neutral detergent fiber (NDF)
[[60]19]. The acid–base fractionated hydrolysis method was used to
determine the crude fiber [[61]20].
2.3.2. Determination of Oats Quality Indices
The relative feeding value (RFV) was calculated as described by Gao
[[62]21] using the following formula:
[MATH:
RFV = DMI (%DW) × DDM (%DW)/1.29 :MATH]
(1)
where DMI represents the dry matter intake, and DDM indicates the
digestible dry matter;
[MATH:
DMI=120/NDF :MATH]
(2)
where NDF indicates the neutral detergent fiber;
[MATH:
DDM=88.9−<
mrow>0.779 × ADF :MATH]
(3)
where ADF represents the acidic detergent fiber.
2.3.3. Free Amino Acid-Targeted Metabolomics Determination of Oats
The samples were extracted from storage at −80 °C and accurately
weighed to 60 mg utilizing an electronic balance (AL104, Mettler
Toledo, Greifensee, Zurich, Switzerland). Then, 50 µL of water
homogenate was incorporated into each sample, followed by vortexing for
60 s with a vortex mixer (QT-1, Shanghai Kit Analytical Instrument Co.,
Shanghai, China). A solution of methanol (≥99.0%, Fisher Chemical,
Pittsburgh, PA, USA) and acetonitrile (≥99.0%, Fisher Chemical,
Pittsburgh, PA, USA) (1:1, v/v) was then introduced in a volume of 400
µL, along with 50 µL of a 50 µM internal standard mixture containing 16
isotopes. The samples were incubated at −20 °C for one hour to
precipitate proteins after the mixture was vortexed for 60 s and then
subjected to low-temperature sonication using an ultrasonic instrument
(JP-100, Shenzhen Jiemeng Cleaning Equipment Co., Shenzhen, Guangdong,
China) for two 30 min intervals. Centrifugation was performed at 14,000
rcf and 4 °C for 20 min using a centrifuge (5430R, Eppendorf, Hamburg,
Germany). The resulting supernatant was freeze-dried using a vacuum
freeze dryer (FD-IC-50, Shanghai Bilang Instrument Co., Shanghai,
China) and stored at −80 °C.
Chromatographic separation was conducted using a UHPLC system (1290
Infinity, Agilent, Santa Clara, CA, USA). Standards (≥99.0%,
Sigma-Aldrich, St. Louis, MO, USA) were maintained in an autosampler at
4 °C, with the column temperature set to 35 °C. Mass spectrometry
analyses were conducted using a mass spectrometer (6500/5500 QTRAP,
SCIEX, Framingham, MA, USA) operating in positive ion mode. Quality
control (QC) samples were produced by combining aliquots from all
samples to evaluate data stability and reproducibility. The relative
standard deviation (RSD) for the analyte in the QC samples was under
10%, signifying that the results were stable and reliable.
The distribution diagram of the RSD of free amino acids in the QC
samples is presented in the [63]Supplementary Materials, Figure S1A.
[64]Table S1 and Formula (S1) of the Supplementary Materials present
the relevant standard curves and formulas.
2.3.4. Fatty Acid-Targeted Metabolomics Determination of Oats
The quantitative method was strictly validated following the relevant
standards of the International Organization for Standardization (ISO),
including validation items such as relative standard deviation (RSD),
limit of detection, limit of quantification, and linear range. Detailed
data on the method performance characteristics are provided in the
[65]Supplementary Materials.
Following the gradual thawing of the sample at 4 °C, 60 mg of the
sample was accurately weighed using an electronic analytical balance
(AL104, Mettler Toledo, Greifensee, Zurich, Switzerland) and combined
with 5 mL of dichloromethane (≥99.0%, Sigma, St. Louis, MO,
USA)–methanol (≥99.0%, Fisher Chemical, Pittsburgh, PA, USA) solution
(2:1 v/v). The mixture was thoroughly vortexed, and 2 mL of ultrapure
water was added to wash it. The lower phase of the solution was then
isolated and evaporated to dryness using a nitrogen stream. Following
this, 2 mL of n-hexane was introduced, along with the internal
standard, and the mixture underwent methyl esterification for 30 min.
Following methylation, 2 mL of ultrapure water was introduced, and 2000
μL of the supernatant was aspirated and evaporated under nitrogen.
The residue was re-dissolved in n-hexane, and the supernatant was
transferred into an injection vial for gas chromatography–mass
spectrometry (GC-MS) analysis (Agilent, Santa Clara, CA, USA). The
samples were separated on a capillary column (19091S-433UI: HP-5ms, 30
m × 250 μm × 0.25 μm, Agilent, America) using a gas chromatography
system, with helium as the carrier gas at a flow rate of 1.0 mL/min.
Mass spectrometric analysis was conducted using a triple quadrupole
mass spectrometer (5977B MSD, Agilent, Santa Clara, CA, USA), and the
detection mode was selected ion monitoring (SIM). The QC samples were
produced by combining aliquots from all samples to evaluate data
stability and reproducibility. The relative standard deviation (RSD) of
the analyte in the QC samples was below 10%, signifying reliable and
stable results. The internal standard method was used for quantitative
analysis, with methyl nonadecanoate (≥99%, NU-CHEK Prep, Inc., Elysian,
MN, USA) as the reference material. The calibration process adopted
matrix-matched calibration; that is, a series of concentration standard
solutions was prepared using blank sample matrices. The linear
correlation coefficients (R^2) of the plotted calibration curves were
all greater than 0.99, meeting the requirements of quantitative
analysis.
The distribution diagram of the RSD of fatty acids in the QC samples is
presented in the [66]Supplementary Materials, Figure S1B. [67]Table S1
and Formula (S2) of the Supplementary Materials present the relevant
standard curves and formulas.
2.3.5. Monosaccharide-Targeted Metabolomics Determination of Oats
In this experiment, detection was performed using GC-MS (8890-5977B,
Agilent, Santa Clara, CA, USA) with a triple quadrupole mass
spectrometer, and the detection mode was selected ion monitoring (SIM).
The quantitative method was validated following the relevant standards
of the International Organization for Standardization (ISO), including
validation items such as relative standard deviation (RSD), limit of
detection, limit of quantification, and linear range. Monosaccharide
standards were used as calibration standards, and a series of
concentration standard solutions was prepared using matrix-matched
calibration. The correlation coefficient (R^2) of the calibration curve
was greater than 0.99.
The samples underwent vacuum freeze-drying using a vacuum freeze dryer
(CentriVap LABCONCO, Kansas City, MO, USA). They were ground into a
powder using a ball mill (MM400, Retsch, Haan, North Rhine-Westphalia,
Germany) operating at 30 Hz for 1.5 min. In total, 20 mg of the
resultant powder was then measured into appropriately labeled
centrifuge tubes. A solvent mixture consisting of methanol
(chromatographically pure, Merck, Darmstadt, Hesse, Germany),
isopropanol (Merck, Kenilworth, NJ, USA), and water in a volumetric
ratio of 3:3:2 (v/v/v) was prepared, and 500 μL of this extract was
added to each sample. The samples were vortexed for 3 min and sonicated
at 4 °C for 30 min using a multi-tube vortex mixer (MIX-200, Shanghai
Jingmei, Shanghai, China). Following this, centrifugation was performed
at 4 °C and 12,000 rpm for 3 min using a centrifuge (5424R, Eppendorf,
Hamburg, Germany). In total, 50 μL of the supernatant was aspirated, to
which 20 μL of an internal standard solution at a concentration of 1000
μg/mL was added. The mixture was subjected to nitrogen evaporation and
lyophilization. Subsequently, 100 μL of pyridine methoxide ammonium
salt (99%, Sigma-Aldrich, St. Louis, MO, USA) (15 mg/mL) was added, and
the samples were incubated at 37 °C for 2 h. Following this, 100 μL of
BSTFA (99%, Shanghai Aladdin Biochemical Technology Co., Shanghai,
China) was added, and the incubation continued at 37 °C for another 30
min (Thermo Scientific Forma 311, Thermo Fisher Scientific, Waltham,
MA, USA).
Over 80% of the compounds in the QC samples had coefficient of
variation (CV) values below 0.3, signifying the stability of the
experimental data. Moreover, the proportion of compounds exhibiting CV
values below 0.2 in the QC samples surpassed 80%, indicating
substantial data stability.
[68]Table S2 and Formula (S3) of the Supplementary Materials present
the relevant standard curves and formulas. The parameters of GC-MS are
shown in the [69]Supplementary Materials, Table S3.
2.3.6. Untargeted Metabolomics Determination of Oats
After the samples were slowly thawed at 4 °C, 60 mg of each sample was
weighed using an electronic balance and added to pre-chilled methanol
(≥99.0%, Fisher Chemical, Pittsburgh, PA, USA)–acetonitrile (≥99.0%,
Fisher Chemical, Pittsburgh, PA, USA)–water solution (2:2:1, v/v). The
mixture was homogenized using vortexing with a vortex mixer (QT-1,
Shanghai Kit Analytical Instrument Co., Shanghai, China) and then
subjected to low-temperature sonication for 30 min using an ultrasonic
cleaner (KQ5200E, Kunshan Shumei, Kunshan, Jiangsu, China).
Subsequently, the samples were incubated at −20 °C for 10 min. After
that, the samples were centrifuged at 14,000 rpm for 20 min at 4 °C
using a centrifuge (5430R, Eppendorf, Hamburg, Germany). The
supernatant was collected and dried under a vacuum (FD-IC-50, Shanghai
Bilang Instrument Co., Shanghai, China). For mass spectrometry
analysis, the dried residue was reconstituted in 100 μL of an
acetonitrile–water solution (1:1, v/v), vortexed again, and then
centrifuged at 14,000× g for 15 min at 4 °C. Finally, the supernatant
was injected for analysis.
The samples were separated using a UHPLC system (1290 Infinity LC,
Agilent, Santa Clara, CA, USA) with a HILIC column (ACQUITY UPLC BEH
Amide 1.7 μm, 2.1 mm× 100 mm column, Waters, Milford, MA, USA). The
column temperature was maintained at 25 °C, the flow rate was set at
0.5 mL/min, and the injection volume was 2 μL. The mobile phase
consisted of two components: mobile phase A consisted of a mixture of
water, 25 mM ammonium acetate (≥99.0%, Sigma-Aldrich, St. Louis, MO,
USA), and 25 mM Ammonia solution, while mobile phase B consisted of
acetonitrile (≥99.0%, Fisher Chemical, Pittsburgh, PA, USA). The
gradient elution schedule was 0.5–7 min, and the fraction of mobile
phase B linearly reduced from 95% to 65%. From 7 to 8 min, it declined
linearly from 65% to 40%. From 8 to 9 min, the proportion of mobile
phase B remained constant at 40%. From 9 to 9.1 min, the proportion of
mobile phase B produced linearly from 40% to 95%. From 9.1 to 12 min,
the proportion of mobile phase B was sustained at 95%. The samples were
maintained in an autosampler at 4 °C during the complete analysis
process.
Mass spectrometry analysis was conducted using Q Exactive-series mass
spectrometers (Thermo Fisher Scientific, Waltham, MA, USA), with
detection performed in both the positive and negative electrospray
ionization (ESI) modes. The parameters for the ESI source and mass
spectrometry settings were as follows: nebulizing gas and auxiliary
heating gas 1 (Gas1): 60; auxiliary heating gas 2 (Gas2): 60; curtain
gas (CUR): 30 psi; ion source temperature: 600 °C; and spray voltage
(ISVF): ±5500 V (for both positive and negative modes). The mass
spectrometry acquisition mode was full scan. The primary mass-to-charge
ratio detection range was 80–1200 Da with a resolution of 60,000 and a
scan accumulation time of 100 ms. The secondary level adopted a
segmented acquisition method, with a scanning range of 70–1200 Da; a
secondary resolution of 30,000; and a scan accumulation time of 50 ms.
Raw data were converted to the mzXML format using ProteoWizard,
followed by peak alignment, retention time correction, and peak area
extraction via the XCMS software (version 3.14.0, Scripps Research, La
Jolla, CA, USA). The extracted data underwent initial metabolite
annotation through the combined use of these two tools, with subsequent
structural confirmation referencing the Human Metabolome Database
(HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG).
Fragmentation spectra obtained using liquid chromatography–high
resolution tandem mass spectrometry (LC-HRMS/MS) were utilized, where
MS/MS data assisted in accurate metabolite annotation to ensure the
reliability of identification results. The identified metabolites were
further subjected to data preprocessing, and their functions and
involved metabolic pathways were determined using HMDB and KEGG.
2.4. Determination of Meat Quality
2.4.1. Determination of Carcass Traits
Carcass segmentation involved measuring the thickness of rib meat,
abdominal wall, backfat, and the eye muscle area, as outlined by Ma et
al. [[70]22].
The area of the eye muscle was determined at the cross-section between
the 12th and 13th ribs of the Tibetan sheep during carcass
segmentation. This cross-section was outlined using sulfuric acid
paper, and then, the area was calculated with a 1 cm × 1 cm grid. The
rib thickness was measured as the tissue thickness 110 mm from the 12th
and 13th ribs to the midline of the spine in the Tibetan sheep. The
thickness of the abdominal wall was assessed at a point 127 mm from the
12th and 13th ribs. Furthermore, the backfat thickness was measured as
the fat layer directly above the center of the eye muscle between the
12th and 13th ribs of the Tibetan sheep.
2.4.2. Determination of Meat-Eating Quality
Qualities associated with eating, including pH, color, thawing loss,
cooking loss, cooked meat percentage, and texture, were determined
using the method of Zhang et al. [[71]23] with slight modifications.
Briefly, the pH[45min] and pH[24h] were determined by inserting a
portable pH meter (PHS-3C, Shanghai Leici Instrument Factory, Shanghai,
China) into the meat samples at a depth of 2–3 cm. We calibrated the pH
meter using pH 4.0 and 6.86. An automatic colorimeter (ADCI-60-C,
Beijing Chentaike Instrument Technology Co., Beijing, China) was used
to measure the values of L* (lightness), a* (redness), and b*
(yellowness) on the meat surface. The colorimeter was equipped with a
standard xenon lamp within the close aperture of 8 mm set to Illuminant
D65 with an observer angle of 2°. In addition, the meat samples were
heated in a water bath at a constant temperature of 80 °C until the
internal temperature reached 70 °C. Next, using an iron ruler and a
scalpel, the samples were cut into 3 cm × 1 cm × 1 cm meat columns in
the direction of the muscle fibers. A muscle tenderness meter (RH-N50,
Guangzhou Runhu Instrument Co., Guangzhou, Guangdong, China) was then
used in the direction of the vertical muscle fiber to evaluate the
shear force.
The water-holding capacity was calculated as the ratio of the amount of
water lost by the meat samples to the initial weight of the samples,
which was determined after the meat samples (1 cm × 1 cm × 1 cm) were
subjected to a pressure of 350 N by a water-holding capacity tester
(RH-1000, Guangzhou Runhu Instrument Co., Guangzhou, Guangdong, China).
The cooked meat percentage was calculated as the ratio of the weight of
the meat samples after being boiled in a water bath at 80 °C for 40 min
to the initial weight of the samples (average weight of 60 g). For
cooking loss analysis, the meat samples (2 cm × 3 cm × 2 cm) were
cooked in a water bath at 80 °C for 30 min, and cooking loss was
calculated as a percentage of the weight change in the samples from the
initial weight of the samples. In the same way, the thawing loss was
calculated as the ratio of the weight of the meat samples after being
unfrozen in a refrigerator at 4 °C for 12 h to the initial weight of
the samples (average weight of 30 g). Next, a texture profile analysis
(TPA) analyzer (TA.XTC-18, Shanghai Baosheng Industrial Development
Co., Shanghai, China) was used to measure the hardness, elasticity,
adhesion, cohesion, and chewiness of the samples (1 cm × 1 cm × 1 cm).
The probe model utilized was TA3/100, while the fixture model was
TA-RT-KIT.
2.4.3. Determination of Meat Sensory Evaluation
This study references the standards ISO 13299:2016 (en) and ISO 5492