INFLUENCE OF BEEF MARBLING SCORE ON QUALITY INDICATORS DURING MEAT AGING

КЛЮ ЧЕВЫ Е СЛО ВА: говядина, ст епень м рам орност и, созреван ие, органолепт ические, м икрост рукт урные показат ели, усилие резания В эксперименте использовали образцы говядины степеней мраморности «умеренная» и «хорошая» (п=4), половину образцов упаковывали в полимерную пленку под вакуумом, половину оставляли в неупакован­ ном виде. Созревание проводили в камере хранения охлажденного мяса при температуре 2 ± 1 °С и относи­ тельной влажности не более 90 % в течение 4, 16 и 28 суток. В процессе созревания говядины исследовали величину pH, органолептические показатели уровня качества и свежести, микроструктурные характери­ стики, усилие резания. По органолептическим показателям свежести вне зависимости от степени мрамор­ ности на 28 сутки говядина в неупакованном виде имела признаки несвежего мяса, в упакованных образ­ цах отклонений не установлено. Органолептическая оценка уровня качества свидетельствовала о высоких показателях вареного мяса и бульона в течение всего времени созревания для упакованного мяса и в те­ чение 16 суток для неупакованного вне зависимости от уровня мраморности. В результате гистологи­ ческих исследований установлено, что с увеличением срока созревания говядины усиливался характер деструктивных изменений тканей. Образцы упакованной говядины, как с мраморностью «хорошая», так и «умеренная», на 16 и 28 сутки соответствовали второму и третьему этапу созревания мяса, изменения неупакованной говядины на соответствующие сутки созревания были менее выражены. Результаты иссле­ дования усилия резания свидетельствовали о том, что в течение всего наблюдаемого периода созревания происходило постепенное снижение усилия резания.


Introduction
Due to the development of beef production in Russia, the culture of meat consumption has also been developing in recent years.One of the important processes that have spread in our country is a prolonged beef aging.Due to prolonged aging, it is possible to obtain a high-quality product with excellent sensory indicators and taste properties.Since prolonged aging is a pro cess that requires high energy consumption and strict adherence to all condition requirements, usually it is used for raw materials of the highest price category, in particular high-quality marbled beef [1,2].
The production of such beef is possible only with specialized cattle meat breeds: predominantly Aberdeen-Angus, Hereford, Wagoo (most common in Japan, but also grown in other coun tries) and others.This is due to the fact that the marbling score is primarily determined by genetic factors: a number of genes are associated with it, but GG genotype of the GH gene is the most desirable for marbling.For the production of high-quality beef, bulls and heifers are used of 8 months to 2 years old, as well as steers of 8 to 30 months old [3].
Also, to obtain beef with a high marbling score, even from ge netically predisposed breeds, high-energy (grain) finishing diet is required for at least 100 days.Feeding diets may be different depending on the region, so corn, oats, barley and other cereals can be used.
The generally accepted world practice is a high culture of high-quality beef production and the introduction of «From Field to Fork» traceability system -from the selection of ani mals and their antemortem productivity assessment to pack aged products.
Aging is an important process in meat production for cook ing, which occurs when carcasses (half-carcasses, quarters, cuts) after slaughter are exposed to certain conditions.As a result of aging, meat tenderness, juiciness, specific pleasant taste and fla vor increase.
Meat aging is the initial stage of autolysis, so it is determined by the activity of the endogenous enzymes.It is important not to allow the growth of undesirable microflora, the enzymes of which may also cause lysis of cellular structures in animal tis sues and breakdown of nutrients.
Immediately after slaughter, the meat is hot, and then the temperature of muscles decreases with the onset of rigor mor tis.The development of rigor mortis stops with the beginning of meat aging.
During aging, complex biochemical processes occur, leading to a change in physical and chemical properties, structural and mechanical properties and sensory indicators of meat [4,5,6].
Meat obtained immediately after slaughter with a tempera ture of at least 35 °C at any point of measurement is considered «hot».Hot meat is characterized by a soft muscle texture.Its ability to hold the moisture contained in it is as high as possible.Flavor and taste are poorly expressed.The pH value of hot meat is 6.8 to 7.3.
Rigor mortis in beef (for meat in half-carcasses) begins about 3 hours after slaughter.The development of rigor mor tis at a temperature of 0 to 4 °C reaches its maximum by 24 hours from the moment of slaughter.Meat in the state o f rigor mortis is characterized by increased toughness, minimal wa ter-holding capacity, and the lowest pH values (for beef, as a rule, the pH decreases to 5.5), which deteriorates its sensory indicators.
Approximately 48 hours after slaughter, rigor mortis passes into the resolution stage.During normal autolysis, pH slightly increases, and water-holding capacity rises too.At the same time, aging process begins, which improves the sensory indica tors of meat, including texture, taste and flavor.
The rate of meat aging depends on both endogenous factors, 1. e. type of muscles, the amount of lactic acid and glycogen, and exogenous factors, the main of which are the ambient tempera ture and the conditions of heat and mass transfer.
Beef (in half-carcasses) is considered aged after its storage at a temperature of 0 to 4 °C at least for 5 to 7 days.By this time, the development of autolysis leads to significant beneficial changes in the quality of meat, i.e. decrease in toughness, improved flavor and taste.However, meat aging may take long time depending on the desired degree of sensory indicators.For example, a number of studies have shown that beef aging for up to 10 to 14 days is advis able, while with longer period no further improvement in sensory indicators is noted.Later studies showed that a period of beef ag ing up to 25 to 30 days or more may be recommended.
It is important to understand that the result of changes in sensory indicators during meat aging depends not only on the duration of aging itself, but also on the nature of the autolysis.The best aging result is possible only for beef obtained from animals maintained in proper conditions, with the correct and humane handling until the slaughter.Deviations from the good manufacturing practices may cause significant changes in bio chemical processes that occur in meat during autolysis, both in time and in nature, as well as in the final result leading to con sumer dissatisfaction.
In industrial conditions, meat aging (in half-carcasses) is carried out at 0 to 4 °C and 85 % relative humidity for at least 3 days.Then it is transferred for sale or production of meat foods.Under these conditions, the weight loss of half-carcasses is min imal, i.e. not more than 3 % of hot carcass weight.
In foreign practice, to obtain beef with distinctive sensory in dicators, the following methods of its prolonged aging are used: □ dry-aging -in half-carcasses, quarters and bone-in cuts in unpackaged form, suspended or on shelves (racks) at 0 to 4 °C and relative humidity not more than 75 %; □ wet-aging -in bone-in and boneless cuts, in packaged form (with the use of impermeable film materials, under vacuum) at a temperature of 0 to 4 °C; the relative humidity does not matter, since the contact with air is excluded; packaged meat is laid in one row on shelves or racks.Beef aging may be carried out for a prolonged time provided that hazards associated with microbiological risks are excluded: for the first method, by limiting humidity and creating a dried surface meat layer of significant thickness; for the second meth od, by packaging, which prevents the development of aerobic m i croflora.Typically, meat aging in industrial conditions is carried out in cooling rooms.By the end of aging, meat is transferred for sale, where it continues to be stored under similar conditions.
Literature data contain foreign publications on meat quality after prolonged aging, however, they are few and refer to meat obtained from certain breeds of cattle and certain conditions of their growth and slaughter specific to other countries.In this re gard, the study of the quality of Russian meat after prolonged aging is of scientific and practical interest.
The objectives of the study were: □ on the basis of complex studies, to draw a conclusion about the consumer properties of beef with various marbling scores; □ to study the change in sensory indicators, structural and m e chanical properties of beef with various marbling scores dur ing the aging process; □ to determine the optimal aging period for beef with various marbling scores in a packaged and unpackaged form.

M aterials and m ethods
The experiment on aging was carried out on in-bone dorsal beef cut (n =4), obtained during the cutting of cattle carcass es from Aberdeen-Angus breed (black angus) at the age of 18 The pH was measured by a potentiometric method using Tes to 205 portable pH meter.During the measurements, the elec trode was immersed in the muscle tissue to a depth of at least 3 cm.The final result was the mean of three single measurements; the difference between the limiting values of three measure ment results did not exceed 0.15 pH units.
Determination of muscle tissue structural and mechanical characteristics was carried out using AGS-X series Shimadzu test machine designed to measure shear force, surface and compres sion parameters.All measurements were carried out in four rep licates.The final result was the mean with a significance level P > 0.95.
The study of sample microstructure was conducted in accor dance with GOST 19496-2013 «Meat and meat products.His tological examination method».A piece of 2 x 1.5 x 0.5 cm was taken from each sample and placed in a 10 % aqueous solution of formalin for 72 hours at room temperature.Then piece was washed under cold running water for 12 hours.The washed ma terial was put into trays, filled with 25 % gelatin and placed in a thermostat at 35 °C for 12 hours to impregnate.
The resulting piece was placed in a freezing chamber of MIKROM-HM525 microtome-cryostat (Carl Zeiss, Germany) on object table .Freezing was conducted at a temperature of -2 0 °C for 15 to 20 minutes and sections were made with a thickness of 20 pm.
The sections were stained with Ehrlich's hematoxylin and dyed with freshly prepared 1 % aqueous-alcoholic eosin solu tion, and then placed under cover slips in glycerin-gelatin.
Histological specimens were studied and photographed us ing the Axiolmaiger A1 microscope (Carl Zeiss, Germany) using connected AxioCam MRc 5 video camera.Image processing was performed using AxioVision 4.7.1.0digital image analysis sys tem adapted for histological studies.
Sensory evaluation and quality assessment of samples after cooking were carried out using 9-point scale in accordance with GOST 9959-2015.Beef freshness evaluation was carried out in accordance with GOST 7269-2015.

Results and discussions
As a result of marbling evaluation, it is established that the samples were characterized by marble scores «moderate» and «good» (Table 1).Unpackaged samples were labeled as 1-1 (mar bling score «good») and 2 -1 (marbling score «moderate»), while samples packaged under vacuum in polymer film were labeled as 1 -2 (marbling score «good») and 2 -2 (marbling score «moderate»).
Throughout the aging period, the pH of beef remained stable (Table 2).This was due to the fact that significant pH changes were observed in beef in the first 4 days after slaughter.Then, during the further storage of meat, the pH remains stable, and slight increase may occur as a result of putrefaction and the ac cumulation of protein decomposition products having alkaline properties.The stability of pH during the research indicates the normal development of meat aging process.The stability of pH during aging process is also noted by other researchers, e.g.Hulankova et al. [4].
Table 2 The pH of the samples during the aging process Aging period, Samples days According to the sensory indicators of freshness (surface appearance and color, the condition of muscles on the section, texture, odor, fat condition, transparency and broth flavor), beef with various marbling scores in packaged and unpackaged form predominantly considered as fresh meat.However, regardless of the marbling score, the beef aged in unpackaged form had devia tions in sensory indicators on day 28.It was noted that meat on the section was less dense, less elastic; the fovea formed with the finger pressing straightened more slowly; there were also sour and not typical for fresh meat odor and slightly turbid broth.
Analysis of data from beef sensory evaluation during the aging process showed that, on days 4 and 16, after the boiling, the overall quality assessment according to the 9-point scale for vacuum-packaged beef with marbling score «good» (sample 1 -2 ) and «moderate» (sample 2 -2 ) was 8 points («very good») (Table 3), while on day 28, there was an increase in tenderness from 8 to 9 points.Overall sensory evaluation of broth resulted in 8 points («very good») (Table 4).
On days 4 and 16, overall beef quality evaluation after cook ing according to the 9-point scale for unpackaged beef with mar bling score «good» (sample 1 -1 ) and «moderate» (sample 2 -1 ) resulted in 8 points («very good») (Table 5), while on day 28, there was a decrease in tenderness from 8 to 7 points.On days 4 and 16, overall sensory evaluation of broth resulted in 8 points «very good», but on day 28, it decreased to 5 points (Table 6).
The results obtained are in agreement with the works of oth er authors.Thus, Berger et al. [7], studied three different aging methods -in vacuum-packaged form, in air-permeable package (combined method), in unpackaged form -and noted that beef aged for 7 days after slaughter in unpackaged form and in com bined package had higher sensory indicators than aged in pack aged form.In the samples aged in combined package, less mois-    ture loss was noted than in samples aged in unpackaged form.Our experiment did not investigate aging in combined package; this is the object for further research.

Quality evaluation of broth according to the 9-point scale
Similar results were obtained by American researchers Colle et al. [8].Their work was aimed at prolonged aging of steaks from M. biceps femoris and M. semimembranosus.Aging was car ried out for 2, 14, 21, 42, and 63 days.In all cases, tenderness, juiciness, and sensory indicators improved.They also conducted the study [9] on the quality of steaks from M. gluteus medius and M. longissimus lumborum for 2, 14, 21, 42, and 63 days.Improve ment of sensory indicators in all samples was noted.The data obtained indicated that for steaks from M. gluteus medius shear force slightly decreased throughout the aging period, whereas for steaks from M. longissimus lumborum a decrease was noted already on day 21 of aging.
The results of histological studies to determine the structure of beef samples with various marbling scores during the aging process are presented in Tables 7 and 8 and in Figures 1 to 12 (ag ing in unpackaged form) and 13 to 24 (aging in packaged form).
Histological examination of sample structure on day 4 of ag ing showed that all samples (1 -1 , 1 -2 , 2 -1 , and 2 -2 ) have simi lar microstructural indicators.In the cross section, the muscle fibers are characterized by a densified packing in bundles of the first order.The shape of the fibers is polygonal or slightly round ed.Endomysium interlayers are well pronounced; the boundar ies between individual muscle fibers are well defined.Diameter of muscle fibers is 55 to 60 pm.
In the longitudinal section, muscle fibers are predominantly straightened, but there are also some fibers of curly form (the stage of rigor mortis resolution).Cross-striation is well pronounced.In some fibers, there are areas with longitudinal striation indicat ing the zones of contraction.The nuclei in muscle fibers are well colored, oval in shape and located directly under the sarcolemma.
Perimysium interlayers are curly, tightly adjacent to bundles of muscle fibers.The nuclei in the connective tissue layers are clearly defined on the slide.Between the bundles of muscle fi bers, in perimysium, there are areas of adipose tissue with a typi cal histological structure.Histological examination of sample structure on day 16 of aging showed that, in samples of unpackaged beef 1 -1 and 2 -1 , the following microstructural changes were observed.Along with the pronounced cross-striation, in some muscle fibers there are sites with smoothened striation.The color of fibers is not uniform.The nuclei in muscle fibers are shady, worse defined on the slide in comparison with the corresponding samples on day 4 of aging.In muscle fibers, there are multiple fissured trans verse integrity disorders, single ruptures and fragmentation.There are areas with sarcolemma separation, as well as with the destruction of fiber internal structure (local degradation of sarcomeres).There is a loosening o f perimysium interlayers.Sample microstructure corresponds to fresh raw meat of non prolonged storage at the beginning o f the second aging stage (Figures 5 to 8).In samples of packaged beef 1 -2 and 2 -2 , the changes in tis sue structure are more pronounced.In muscle fibers, there are large areas with smoothened striation.The nuclei are shady, and in some fibers, are completely degraded.In muscle fibers, there are multiple fissured transverse integrity disorders and ruptures, fragmentation with formation of fine-grained protein mass be tween fragments.There are long areas with sarcolemma separa tion, as ■well as with the destruction of fiber internal structure (local degradation of sarcomeres).There is a loosening of peri- The resu ltso f shear force ntudy lor samples in packaged form oC :. mysium interlayers, and in some areas, they are separated from muscle fibers.Sample microstructure corresponds to fresh raw meat at the middle of the second aging stage (Figures 17 to 20).Histological examination of sample structure on day 28 of aging showed that destructive changes were intensified in tis sues of all samples.

Results of histological examination of samples in unpackaged form
In unpackaged samples 1-1 and 2 -1 , cross-striation only partly present throughout the muscle fiber, and sometimes is smoothened.The color of the fibers is faded, and not uniform.The nuclei in muscle fibers are shady or completely degraded.On day 16, an increase in ruptures and fragmentation of mus cle fibers was observed in comparison with the corresponding samples, with the formation of fine-grained protein mass b e tween fragments.There are areas with sarcolemma separation and degradation, as well as with the destruction of fiber internal structure (local degradation of sarcomeres).There is a loosening of perimysium interlayers, and in some areas, they are separated from muscle fibers.There are foci of microflora.The nuclei are worse defined on the slide.Sample microstructure corresponds to raw meat of ambiguous freshness category at the end of the second aging stage (Figures 9 to 12).
In samples 1 -2 and 2 -2 , in most muscle fibers, the striation is smoothened.The nuclei are shady, and in some fibers, com pletely degraded.In muscle fibers, there are multiple ruptures and fragmentation with separation of fragments and the forma tion of fine-grained protein mass between them.There are long areas with sarcolemma separation, as well as with the destruc tion of fiber internal structure.The disintegration of individual fragments into myofibrils is detected, and myofibrils are de graded into sarcomeres in the form of grained mass sometimes confined to endomysium.There is a loosening of perimysium interlayers and separation from muscle fibers.Changes in tissue structure correspond to fresh raw meat of non-prolonged stor age at the third aging stage (Figures 21 to 24).
As a result of histological studies, it was found that, with the increase in the duration of beef aging, the nature of destruc tive changes in tissues is enhanced.On days 16 and 28, samples of packaged beef, both with marbling score «good» (1 -2 ) and «moderate» (2 -2 ), corresponded to the second and third stage of meat aging in accordance with GOST 19496.The raw material remained fresh throughout 28 days.
The results of shear force study (Tables 9 and 10) showed that during the entire observed period of aging, a gradual decrease in shear force occurred indicating meat softening.However, in some cases, shear force at a later stage of aging was higher than at the previous one.This may be explained by the properties of marble meat structure, in the muscular tissue of which there are multiple inclusions of adipose tissue.In average, on days 4 and 16 of aging, the toughness of vacuum-packaged beef was lower than in unpackaged beef, indicating greater efficacy of meat ag ing in a packaged form.However, at later stages of aging, the dynamics of decrease in shear force of packaged beef declined.
A.N. Lepper-Blilie et al. [10] also revealed an inverse correla tion between shear force and aging period.According to these data, the degree of shear force decrease depended neither on carcass part, nor on aging method, nor on the marbling score.However, in the work of Yuan H. Brad Kim et al. [11], there is evidence that shear force after 17 days of aging was lower in packaged samples than in unpackaged indicating faster aging in packaged form.L. Clay Eastwood et al. [12] also noted that shear force depended, in part, on the type of aged product, e.g. for samples in cuts, shear force was lower than for samples in the form of semi-finished products (steaks).Fumiko Iida et al. [13] found that shear force and tenderness of beef with a high marbling score stabilized during 20 days of aging, while the im provement in taste and flavor continued up to 60 days.

Conclusion
The aging method has a great influence on the quality in dicators of beef (microstructure, sensory indicators, structural and mechanical properties) regardless of the marbling score.
Il'ya V. Kozyrev,* Tat'yana M. Mittelshtein, Tat'yana G. Kuznetsova, Viktoriya A. Pchelkina, Kiril I. Spiridonov, Andrey B. Lisitsyn V.M .G orbatov Federal R esearch Center for Food System s o f R ussian Academ y o f Scien ces, Moscow, Russia К Л Ю Ч Е В Ы Е С Л О В А : In th e experim ent, b e e f sam ples were used w ith th e m arbling scores «m oderate» and «good» (n = 4).H alf o f the beef, m arblin g score, aging, sam ples were packaged in a plastic film under vacuum , w hile th e rest ones were left unpackaged.Aging was carried sen sory indicators, m icrostru ctural ou t for 4, 16, and 28 days in a storage cham ber at a tem perature o f 2 ± 1 °C and a relative hum idity of no t m ore than indicators, s h e a r fo r c e 90 %.During b e ef aging, pH, sensory indicators o f quality and freshness, m icrostructural indicators, and shear force were evaluated.Regardless o f th e m arbling score, on day 28, sensory indicators o f unpackaged b e e f had the signs o f n o n-fresh m eat, while th e packaged sam ples had no deviations.Regardless o f the m arbling score, sensory evaluation indicated th e high quality o f boiled m eat and broth during th e entire aging period for packaged b e e f and during 16 days for unpackaged beef.

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samples months fed with grain diet during 200 days.The slaughter and the primary processing of carcasses were carried out at Bryansk Meat Company LLC (Miratorg Agribusiness Holding), Bryansk Region.The cuts were removed 48 hours after the slaughter.Beef aging was carried out for 28 days in packaged and unpackaged form in a cold storage chamber at a temperature of 2 ± 1 °C and a relative humidity of not more than 90 %.Marbling score was determined visually according to GOST 33818-2016 «Meat.High quality beef.Technical condi tions».

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Picture 13.Cross section Picture 15.Cross section 4 Picture 14. Longitudinal section Picture 16.Longitudinal section 16 28 Picture 17. Cross section Picture 19.Cross section Picture 18. Longitudinal section Picture 20.Longitudinal section Picture 21.Cross section Picture 23.Cross section Picture 22. Longitudinal section Picture 24.Longitudinal section The functional state of muscle tissue is quite uniform There are individual transverse micro-ruptures, single sites with local sarcomere degradation.Sarcolemma deterioration, myofibril de struction of and muscle fiber ruptures was not detected.Sample microstructure corresponds to fresh raw meat at the first aging stage (Figures 1 to 4 and 13 to 16).