Nutritional, physicochemical, textural and sensory characterization of horsemeat patties as affected by whole germinated green buckwheat and its flour

ABSTRACT

This work investigated the biological efficacy of germinated green buckwheat and its flour (GGBF) as an improver of the quality, storability, and safety of mixed horsemeat and chicken (thighs) patties. The addition of germinated green buckwheat and its flour (GGBF) into the patties improved (p ≤ .05) the protein and fat contents, cooking yield, moisture and fat retention, total phenolic content, and DPPH radical scavenging activity. The addition of GGBF resulted in a lower L* value, whereas the a* and b* values were slightly higher, with the storage time exhibiting a preserving effect of green buckwheat sprouts on the color of a product. Also, the sensory evaluation revealed increases (p < .05) in overall acceptability and juiciness scores. Generally, this study identified the antioxidant and textural improvement potentiality of germinated green buckwheat and its flour, which could pave the way for its use as an extender of the shelf life of the patties.

KEYWORDS:

• Green buckwheat
• horsemeat
• mixed meat
• nutritionally rich patties
• quality food

Introduction

In the modern economy, the role of the invention has increased significantly. The meat processing industry needs to present new meat products with high nutritional value and improved taste to sustain market conditions and to offer alternatives to consumers, and it must decrease production costs through the use of non-traditional types of meat and protein supplements. Chopped semi-finished meat products with protein-rich vegetables are very popular with buyers since their qualities are determined by the quality of the raw materials.^[1,2] The meat business has expanded to mass-produce beef and poultry while mostly abandoning horsemeat products.

Horsemeat (adult horse and foal) is valuable not only from an economic point of view since it is one of the most demanded and profitable products on trade today,^[3–5] but horsemeat also has high-value properties such as digestibility, nutritional value, therapeutic and prophylactic values, and dietary and gustatory or gourmet qualities, and even in several ways surpasses beef, lamb, pork and, poultry, as well as olive, cottonseed, sunflower, and other oils, and it can serve as a worthy replacement in the diet. Horsemeat has retained its leadership for centuries, and its popularity is growing due to its unique properties.^[1–6] From a technological point of view, the consumption of horsemeat as the essential raw material in chopped semi-finished products manufacturers recommended. Horsemeat contains complete proteins, fats, carbohydrates, vitamins, and macro and microelements.^[7] Its proteins contain all the essential amino acids. The amino acid composition of horsemeat is ideal for the optimal formula proposed by FAO/WHO. Horse fats are an enormous energy source and beneficial to human consumption due to the relatively high content of unsaturated fatty acids (60.04%), compared to beef fat (27.09%) or yak fat (38.03%).^[8,9] At the same time, in terms of its properties, the properties of horse fat are close to those of olive oil and, to a lesser extent, of cottonseed and sunflower oil, and in these characteristics, it is significantly ahead of poultry, beef, pork, and lamb fat. Horse fat has a choleretic effect and is rich in unsaturated fatty acids (linoleic, linolenic), the absence of which in human food leads to skin diseases.^[10,11] These acids also significantly change the nature of cholesterol breakdown, dissolving it and converting it into compounds that are excreted from the body without difficulty, thereby reducing its level in the blood, preventing its deposition, removing excess, and having a beneficial effect on cholesterol metabolism.^[9]

Green buckwheat flour is a product of sprouted buckwheat, an herbaceous plant of the huge Buckwheat family. The seeds of this cereal culture have been consumed by mankind for more than three thousand years.^[12] Green buckwheat flour is a fairly useful product for the general condition of the whole organism. Since these buckwheat grains do not undergo hydrothermal machining process, they retain the entire range of useful and active substances and vitamins. Buckwheat flour contains dietary fiber represented by cellulose, pectin, lignin, and hemicellulose. Moreover, there are also a lot of minerals. There is very little information on integrating buckwheat sprouts in food preparations. The high nutritional value and various health benefits of buckwheat have attracted food manufacturers to the use of buckwheat and sprout flour incorporated products and sprouted grains have become very popular among the healthy-eating population. The use of plant materials in meat products will give new functional properties, and significantly enrich the finished product with vitamins, minerals, and fiber which increase its nutritional and biological value.^[13] The high benefit of functional ingredients contributes to their unhindered use in the elaboration of new functional products. Adherents of a healthy lifestyle and proper nutrition are undoubtedly familiar with such a product as green buckwheat, although most of the population is skeptical about it, associating the word “green” with immaturity. Green buckwheat is a unique type of cereal that has nutritional value and a supply of nutrients. That is why it is also called live buckwheat. Unlike typical buckwheat, which is present in the kitchen of every house, green buckwheat has retained almost all the valuable substances that nature has endowed it. Few people know, but until the 50s of the last centuries, buckwheat was not processed in the Soviet Union and was delivered to store shelves in green form. The plant owes its nutritional value and other functional properties to an exceptional composition. Buckwheat contains several elements that the human body needs for healthy functioning: the vegetable protein level is up to 15%, which is significantly higher than in other types of cereals, and protein is valuable for healthy muscles, skin, and bones; it contains an amino acid, lysine, which is rare for a plant product, which contributes to the enrichment of valuable blood substances and prevents anemia; it is rich in vitamins of groups B, E, and PP; it contains minerals such as calcium, iron, iodine, copper, magnesium, potassium, zinc, and fluorine: moreover, the minerals in buckwheat are in amounts that the human body needs; it contains antioxidants, which are valuable substances that help the body to fight against free radicals and therefore cancer, and it contains flavonoids, which are pigments that give the substance its color. That is why buckwheat is green; moreover, flavonoids are also responsible for the wellness of internal organs and for slowing down the aging process of the body.

Germination is also significant in buckwheat: it makes the buckwheat more nutraceutical, pharmaceutical, and medicinal. Germination is a complex process in which significant alterations in biochemical, nutritional, and sensory characteristics occur due to the activation of dormant enzymes.^[13] Therefore, sprouts have higher levels of nutrients and a lower amount of compounds which can increase starch and protein digestion. Germinated buckwheat is a vital raw material for food and functional food production, which has better nutritional value than non-germinated buckwheat. The germinated seeds could help prevent and treat various human diseases and can be one of the active components to produce functional foods. The key and fundamental difference is the presence of well-pronounced binding properties of green buckwheat flour, which in many cases makes it possible to be used instead of wheat flour without fear of getting a product that falls apart or crumbles badly.

As far as we know, no information is available on green buckwheat flour as an additive to improve the nutritional quality of meat products. Although germinated green buckwheat has prodigious amounts of phenolic compounds with antioxidant and antimicrobial activities, studies on its application to prolonging the shelf-life of meat products are scarce.^[13,14] Therefore, this study aimed to investigate the effect of the incorporation of non-germinated and germinated green buckwheat flour on the proximate compositions, cooking properties, oxidative characteristics, textural properties, and sensory quality of horsemeat patties.

Materials and methods

Materials

Forty kilograms of boneless horse rump steaks (Musculus semimembranosus) (moisture 75%, protein 21%, fat 2.2%, ash 1.4%) of female Jabe horses (aged 24–28 months fattened for six months), 10 kg of horseback fat from the same carcasses, and 8 kg of chicken thighs (aged four months, moisture 72.5%, protein 23.2%, fat 3.2%, ash 1.15%) were obtained from a commercial market (Semey, Kazakhstan) within an hour of slaughter. Following collection, the cuts were carefully trimmed of all visible fats and connective tissue. Later, the samples were vacuum-sealed and stored at a temperature of −20°C prior to use. Onion (Allium cepa), cabbage (Brassica oleracea var. capitata), salt, green buckwheat (Fagopyrum esculentum) grains were purchased from a local market. The samples were cleaned, washed, and packed separately in polyethylene bags and stored at 4°C in a refrigerator before use. All of the reagents used in this investigation were of analytical grade unless otherwise specified. The experiment was carried out in the laboratories of the Food Production and Biotechnology Department of Shakarim University of Semey, Kazakhstan.

Preparation of germinated green buckwheat (GGB) and its flour

Commercial green buckwheat (Fagopyrum esculentum) was purchased from a local market (Semey, Kazakhstan) and used in this study. The technology features of the germinating process include cleaning grains of impurities, washing, and then immersing in water at 32–33°C for 20 min. The treated grains were germinated on water-soaked urethane foam at 23°C for 24 h in the dark, rinsing thoroughly every 3 h, and, if necessary, wetting the foam with water. After germination, the buckwheat grains were carefully taken out and dried at 50°C for 24 h, blended finely using a laboratory mill, and passed through a < 2 mm (10 mesh) sieve. The germinated green buckwheat flour is white with a greenish tinge and very finely ground. The grain powder was vacuum-sealed with PE/nylon film using a model DZ-260PD vacuum packaging system (Russia) and stored in a refrigerator at 4°C until used. The non-germinated grains used as control were dried at 50°C for 24 h and milled and stored in a vacuum pack at 4°C until use. The antioxidant activity, total phenolic content, proximate composition, and pH of non-germinated and germinated buckwheat flour were measured. Each analysis was determined in triplicate. The technological scheme of buckwheat processing is given in Figure 1. The germination process was continued for seven days.

Figure 1. The technological scheme of buckwheat processing.

Antioxidant activity of flour

DPPH (2, 2-diphenyl-1-picrylhydrazyl) is used to test the antioxidant activity described by authors.^[15] 10 mL of CH₃OH (80%) was mixed with 1 g of germinated green buckwheat flour (GGBF), shaken for 30 min at 25°C, and then centrifuged at 2000 rpm for 15 min. Then, 100 µL of supernatant was added to 3.9 mL of DPPH (0.0025% w/v). The solution was kept at 25°C for 1 h. The absorbance was noted at 517 nm against the methanol by using a UV-1800 Spectrophotometer (Shimadzu UV-1800, 115 VAC, Shimadzu, Japan). Eq. (1)(1) $\mathrm{\%}\mathrm{A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\left(\mathrm{A}\mathrm{b}{\mathrm{s}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}--\mathrm{A}\mathrm{b}{\mathrm{s}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}\right)/\mathrm{A}\mathrm{b}{\mathrm{s}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}\times 100$(1) was used to calculate the antioxidant activity.^[9]

(1) $\mathrm{\%}\mathrm{A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\left(\mathrm{A}\mathrm{b}{\mathrm{s}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}--\mathrm{A}\mathrm{b}{\mathrm{s}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}\right)/\mathrm{A}\mathrm{b}{\mathrm{s}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}\times 100$(1)

Total phenolic content (TPC)

The TPC of the flour was determined according to the work described by Beitâne et al.^[16] with some modifications. Briefly, 10 mL of CH₃OH was mixed with 1 g of flour, stirred at 300 rpm for 1 h, then filtered. Next, 0.5 mL of extract, 0.5 mL of Folin-Ciocalteau reagent, and 7 mL of distilled H₂O were added to a 10 mL tube and kept at 25°C for 8 min. Then, 1.5 mL of Na₂CO₃ (2%) was added to the tube and diluted with distilled H₂O to 10 mL. The mixtures were shaken well and left in the dark at 25°C for 2 h. All absorbance measurements were taken using a UV/Vis spectrophotometer at 765 nm and 1 cm quartz cells. The results were stated as mg gallic acid equivalent (GAE)/100 g of flour.

Preparation of semi-finished meat product with plant components (patties)

Two types of patties were prepared: the control product, with NGGBF (non-germinated green buckwheat flour), and the “Shygys” patty containing sprouted green buckwheat and its flour. For patties elaboration, horsemeat, horse fat, and chicken meat were cut into small cubes and scalded in boiling water in plastic bags for 5 min. The scalded horsemeat (40%), chicken thigh meat (25%), horse fat (7%), sprouted green buckwheat grain (5%), cabbage (4%), and onion (3%) were first ground in a grinder (Moulinex HV6 – ME511H27, France) with 2–3 mm diameter lattice holes. The ingredients were blended with eggs (2%), sprouted green buckwheat flour (2.5%), black pepper (0.3%), salt (1.3%), and water (10%) using a ZB-8 (China) mixer until an evenly mixed texture formed. The prepared mixture was divided by hand into ten patties (50 g each, 8 cm diameter/1.5 cm height), placed on trays, and frozen at −29 to −30°C for 1 h. After checking the finished products, the patties were packed in weights of 500 g (packs of ten patties of 50 g each) in vacuum plastic bags, labeled, and kept at 4°C for nutritional, sensory, and physicochemical analyses The part of the analysis was accomplished with uncooked and grilled patties. The patties were cooked on an electrical grill (Geepas, Model Ggm6001, power: 700 W), at 190°C for 7 min. A digital thermometer (Fantast, China) was used to measure the temperature of the center of patties, which should be 73°C. After cooking, the samples were cooled and stored at 4°C prior to analyses. Triplicate samples from each batch of GGBF-formulated and NGGBF-formulated patties were analyzed on the same day for the quality characteristics. For storage stability studies, uncooked GGBF-formulated and NGGBF-formulated patties were placed separately in polyethylene bags and stored in a refrigerator (4 ± 1°C) for 0, 7, 14, 21 and 23 days. At the specified time intervals, uncooked patties were removed from the refrigerator and cooked as indicated above, and then both uncooked and cooked patties were assessed for quality characteristics. Better product development is always in demand because of the constant stream of new products entering the market. When choosing the components of the patties that were made with a mixture of horsemeat and chicken, careful consideration was given to the construction of a product that would appeal to a large number of consumers who are interested in decreasing their consumption of meat but do not want to give up the flavor, convenience, or familiarity of traditional processed meat products. The key raw material of the combined meat semi-finished product “Shygys” developed in this research study is a grade I horsemeat.

Physical properties (proximate composition, pH, a_w, water holding capacity, and cooking properties)

The chemical composition of non-germinated, germinated green buckwheat flours and raw patties was examined according to standard methods. Moisture, protein, ash, and fat content were determined respectively according to the requirements of ST (State Standard) R 51479–99 (oven drying method), ST 25011–81 using the Kjeldahl method, ST 31727–2012 (muffle furnace), and ST 23042–86 using the Soxhlet method. Fatty acid content was determined according to ST 55483–2013 by gas chromatography, and according to ST 34132–2017. The carbohydrate percentage was obtained as the difference of 100 of the sums of the percentage of moisture, fat, protein, and ash. All determinations were performed in triplicate. The pH values of the flours and uncooked meat patties with and without sprouted green buckwheat were determined with a pH meter (Model 340, Mettler-Toledo GmbH, Switzerland). The pH values of the samples were measured by mixing a 5 g sample with 20 mL of distilled water for 60s in a homogenizer at 8000 rpm (X-1000, USA). Water activity (a_w) was measured by the cryoscopy method with a Hygropalm – AW1 (Rotronic) device. The water holding capacity (WHC) of cooked patties was analyzed according to work done by Serdaroğlu et al.^[17] with some modifications. For evaluation of WHC, 10 g of horsemeat patty was weighed in centrifuge tubes and centrifuged at 12 000 g for 30 min at 4°C. The WHC was expressed as a percentage of bound water (Eq. 2)(2) $\mathrm{W}\mathrm{H}\mathrm{C}\left(\mathrm{\%}\right)=\left({\mathrm{W}}_1/{\mathrm{W}}_2\right)\times 100$(2) .

(2) $\mathrm{W}\mathrm{H}\mathrm{C}\left(\mathrm{\%}\right)=\left({\mathrm{W}}_1/{\mathrm{W}}_2\right)\times 100$(2)

where W₁ – the weight of the sample after centrifugation and W₂ – the weight of the sample before centrifugation. The weight of the ten patties of each batch was recorded at room temperature before and after cooking to calculate the cooking yield, diameter reduction, and change in thickness, fat retention, and moisture retention (given as a percentage).

Color measurement, texture analysis, and sensory evaluation

The color measurements were performed using a Konica Minolta CR-300 Chroma-Meter (Minolta Camera Co., Japan). The “L” value characterizes lightness, and the “a” and “b” values symbolize redness and yellowness respectively. Color measurements of uncooked patties were recorded at seven-day intervals over 23 days of the cold storage test. Texture profile analysis (TPA) was determined with a Texture Analyzer TA-XT Plus (Stable Micro Systems, England) furnished with a d = 50 mm probe at 3.3 mm/s-1. The analysis was performed at 25°C, and patty samples were compressed up to 50% of their original thickness. The TPA test was used to measure multiple parameters such as hardness, springiness, cohesiveness, and chewiness.^[18] The sensory profile of the patties was carried out using the five-point system, ranging from 1 (extremely disliked) to 5 (extremely liked) according to ST R 9959–2015. Twenty-five non-trained potential consumers (students and staff of the Shakarim University of Semey) assessed the indicators of taste, color, flavor, texture, and overall acceptance. The patties were prepared according to the procedure described earlier. Each sample was coded with random numbers of two digits. The protocol for sensory analysis was approved by the Ethics Committee (Shakarim University of Semey, Semey, Kazakhstan – protocol #126). Bread and water were provided to clear the mouth between the samples. Patty samples were fried according to the method described in 2.2 and were served in random order at 40°C.

Statistical analysis

All the data analyses were carried out in triplicate and the results were presented as the means and standard deviations of three different trials using Statistica 12.0 (STATIS-TICA, 2014; StatSoft Inc., Tulsa, OK, USA). Analysis of variance (one-way ANOVA) was carried out on the chemical analyses, whereas two-way ANOVA was used on sensory analysis, considering formulation and panelist as independent variables. The alterations were interpreted to be statistically important at p ≤ .05.

Ethics statement

The studies involving human participants were reviewed and approved by the local Ethics Committee (Shakarim University, Semey, Kazakhstan – protocol #126). The participants gave their written agreement to participate in this study. All suppliers were informed before the study and enrolled in the project.

Results and discussion

Horsemeat is becoming particularly appreciated on the market due to its characteristic share of proteins, fat, and glycogen. For nutrition-demanding consumers, the relation between essential amino acids and fatty acids in horsemeat and the antioxidant properties of plant products are in great favor.^[7,8,14] Horsemeat is easily digestible due to its high content of proteins, vitamins, and iron, and its small proportion of fat. Like other animal protein sources, the proteins from horsemeat have great nutritional value. The smaller content of connective tissue in horsemeat makes it easier to digest than beef or mutton, and the proteins from horsemeat are even better than proteins from pork, beef, or veal. Due to its characteristics, it is recommended for sick people who have problems with cholesterol and heart conditions and those who are anemic, owing to its high iron content. Horsemeat represents a specific and valuable food of animal origin.^[3–5,7]

The sprouting of seeds has been known for a very long time. Lately, sprout intake has increased mainly because of its nutritional value: it is an excellent source of proteins, fibers, vitamins, minerals, and various bioactive compounds. Germination enhances digestibility by increasing the bioavailability of minerals and phenolic content, which can enrich antioxidant activity.^[19] Given their peculiar characteristics, such as unique color, rich flavor, and appreciable content of bioactive substances, sprouts can enhance the sensorial properties of a wide variety of high-quality products. Moreover, sprouting is a simple and inexpensive process that can be done without sophisticated apparatus, has a quick production cycle (two to three weeks at most), takes up very little space in greenhouse production,^[20] and delivers a large amount of product.

Physico-chemical properties of non-germinated and germinated green buckwheat flours

The results of chemical composition, antioxidant activity, pH, and total phenolic content of non-germinated green buckwheat (NGGB) and germinated green buckwheat flours (GGBF) are presented in Table 1. The obtained results indicate that the moisture, protein, ash, and fibers content were much higher in the germinated buckwheat flour than in the non-germinated sample. The protein content results are in good agreement with values provided by Hung et al.^[21] (19.35 ± 2.14%) and Sturza et al.^[13] (18.75 ± 0.56), respectively. A decrease in fat content after germination is possibly due to the increased activity of lipolytic enzymes during germination.^[22]

Table 1. Physico-chemical properties of NGGBF and GGBF.

	Parameter	NGGBF	GGBF	p-Value
Chemical composition	Moisture, %	9.20 ± 0.25	11.14 ± 0.18	**
	Ash, %	2.09 ± 012	1.52 ± 0.32	**
	Protein, %	12.21 ± 0.22	18.79 ± 0.57	**
	Fat, %	3.36 ± 0.10	2.53 ± 0.22	**
	Fiber, %	7.28 ± 0.23	9.67 ± 0.11	**
	Carbohydrates, %	68.75 ± 0.02	63.05 ± 0.54	*
Antioxidant activity	TPC	200.35 ± 1.50	397.52 ± 1.50	**
	DPPH, %	63.25 ± 1.03	87.83 ± 1.03	**
pH-value		6.25 ± 0.02	5.36 ± 0.16	**
aW		0.43 ± 0.04	0.35 ± 0.05	**
Color	L*	90.54 ± 0.35	97.48 ± 0.22	*
	a*	0.45 ± 0.45	−0.15 ± 0.02	**
	b*	7.25 ± 0.35	8.43 ± 0.08	*

L*, a*, and b* refer to lightness, redness and yellowness, respectively. All values are mean ± standard deviation of triplicates. p-value: ** (p < 0.01), * (p < 0.05)

The water activity and pH values were significantly lower, as observed in the powders. The decrease in the carbohydrate content is also following the results reported by Hung et al.^[21] In addition, the results obtained showed increased antioxidant activity and TPC in the germinated green buckwheat flour after seven days of germination. This can be explained by increasing hydrolytic enzyme activity due to the germination process.^[21] The high level of polyphenolic components in buckwheat shows a high level of antioxidant activity of germinated green buckwheat. The content of the phenolic compounds after germination showed 3.97 mg/g, which is possibly due to the breakdown of the cell wall during germination. The color characteristics of additives influence consumer acceptability and therefore play a significant role in the quality of additives. Germination altered the color characteristics of the flour. The L* and b* values of flour of germinated green buckwheat seeds compared to flour from non-germinated sources slightly increased. However, the a* value was decreased by germination. Changes in the color parameters of flour produced by germination are complex and affected by different features such as variation, germination methods, protein content, carbohydrate content, and bioactive component activities.^[23]

Chemical composition of uncooked horsemeat patties incorporated with NGGBF and GGBF

Table 2 indicates the results of the proximate composition (protein, moisture, ash, and fat) of the uncooked horsemeat patties with the addition of germinated green buckwheat flour (GGBF). There were visible differences in the results obtained between patties with added non-germinated and germinated buckwheat flours. The moisture content was higher (p < .05) for uncooked patties with GGBF (71.56%) as compared with the control sample (65.32%). This is probably due to the vegetables and sprouts of green buckwheat incorporated in the patty’s formulation. According to Ahmad et al.,^[24] the incorporation of dietary fiber increases the moisture content and improves emulsion stability in chicken sausages. The low protein content in NGGBF leads to a lessening in the protein in the control sample. Similar results were reported by authors,^[15] where buckwheat flour showed lower protein content in the final product. A decrease in fat content in the “Shygys” patties is probably due to the incorporation of the germinated green buckwheat and its flour as well as the vegetables. These findings are in good agreement with the results of Yang et al.^[25] and Ahmad et al.,^[24] that the incorporation of hydrated oatmeal and tofu (15%) and different vegetables lower the fat content.

Table 2. The chemical composition of raw horsemeat patties “Shygys” with NGGBF and GGBF.

Parameter	With NGGBF	With GGBF (“Shygys” patties)	p-value
Moisture	65.32 ± 1.5	71.56 ± 1.2	**
Protein	9.5 ± 0.15	18.85 ± 0.12	**
Fat	9.7 ± 0.3	5.63 ± 0.3	*
Ash	2.58 ± 0.19	2.60 ± 0.15	ns
Carbohydrate	14.25 ± 0.13	14.46 ± 0.12	*
pH	5.95	5.98	ns
aw	0.934 ± 0.005	0.920 ± 0.005	ns

All values are mean ± standard deviation of triplicates. p-value: ** (p < 0.01), * (p < 0.05), ns – not significant

The a_w of the control and final test patties showed only a slight change with the addition of GGBF. The authors^[26] reported that the addition of buckwheat flour (5%) decreased a_w and pH during 21 days of storage in the production of salami sausages. The pH values of uncooked patties were 5.95 and 5.98 for NGGBF and GGBF added samples respectively. The GGBF and GGB sprouts inclusion did not cause any visible changes in the pH value of horsemeat patties (Table 2). Similarly, authors^[27] have reported that the incorporation of sprouted wheat flour did not affect the pH of beef burgers.

Effect of germinated green buckwheat flour on the oxidative characteristics of uncooked horsemeat patties during storage

The antioxidants incorporation into meat products results in minimizing rancidity, increasing the shelf-life of food products, conserving nutritional value, and delaying the development of toxic oxidation products.^[14,15,21] The addition of GGBF and sprouts into patties would help provide the benefits of the high antioxidant properties of germinated buckwheat. The number of phenolic compounds in buckwheat, which increases during germination, could be a good way of accumulating phenolic compounds in the human body.^[28] Polyphenols have attracted much attention in diet due to their numerous benefits for human health. The green buckwheat sprouts incorporation and their flour into horsemeat patties would help to deliver the benefits of high antioxidant properties. Table 3 shows the total phenolic and DPPH radical scavenging activity of uncooked horsemeat patties incorporated with GGBF and NGGBF during cold storage (4.0 ± 1.0°C). Based on the results, the DPPH values in horsemeat patties with germinated green buckwheat were higher than those with non-germinated green buckwheat flour, indicating that the incorporation of GGBF, as well as sprouts, improved the antioxidant effects in horsemeat patties. This rise could be due to the substantially high DPPH activity of the GGBF (87.23%) shown in Table 1. These findings are in good agreement with Sturza et al.,^[13] who detected that the addition of sprouted buckwheat flour (30%) into wheat flour in buns increased total antioxidant activity by 59.04% compared with non-sprouted buckwheat flour. Similar results have shown that the incorporation of vegetables^[24] and various health-related ingredients such as chia and quinoa resulted in a progressive increase in DPPH radical scavenging activity in meat products.^[29,30] Alvarez-Jubete et al.^[31] confirmed a higher DPPH radical neutralizing activity of buckwheat seed extracts compared to amaranth, quinoa, and wheat.

Table 3. Oxidative characteristics of raw horsemeat patties formulated with NGGBF (control) and GGBF (test) during storage at 4°C (± 1.0°C).

Formula	Storage days					p-value
	0	7	14	21	23
	Total phenolic (% mg/100 g)
Control	205.08 ± 0.255	200.96 ± 610	194.21 ± 0.303	186 ± 0.774	175 ± 0.305	**
Test	380.33 ± 0.612	377 ± 0.053	373.93 ± 0.140	368.34 ± 0.030	360.72 ± 0.275
	DPPH (%)
Control	61.21 ± 0.583	57.62 ± 0.678	56.61 ± 0.770	54.71 ± 0.300	52.97 ± 0.482	**
Test	89.95 ± 0.673	87.25 ± 0.503	84.32 ± 0.483	80.66 ± 0.562	74.54 ± 0.763

Values are means of triplicate samples (±SE). p-value: ** (p < 0.01)

The polyphenol content was closely related to the antioxidant capacity; cooking caused partial loss of polyphenols and, therefore, the antioxidant capacity. It was observed that the patties with GGBF and sprouts recorded the highest phenolic content along with antioxidant capacity compared to the control sample (NGGBF-incorporated patties). The GGB and its flour addition in horsemeat patties gradually increased the TPC. The enhancement of TPC could be due to the high amount of phenolic in sprouts (Table 1). This data showed that the radical scavenging effect of the extract of the patties is acceptably correlated with the total amount of phenolic compounds. Boo et al.^[32] also presented a positive correlation between the number of phenolic compounds and the DPPH free radical scavenging effect in extracted samples. Thus, the high level of antioxidant activity in horsemeat patties was attributed to the high level of TPC in GGBF. Our results are in accordance with results obtained by Devatkal and Naveena^[33] on ground goat meat. The TPC and antioxidant activity slowly lessened in both samples during the 23 days of storage. The DPPH radical scavenging activity increased until day ten, after which it declined as the storage time continued to 23 days. However, the DPPH and TPC levels were still high after 23 days of storage compared to patties with NGGBF. Decomposition of TPC during storage, maybe be the cause of the decrease in TPC level. Wagh et al.^[34] reported that phenolic compounds act as an antioxidant due to their redox properties which play a significant role in absorbing and neutralizing free radicals. As a result, radical scavenging decreased due to the relationship between TPC and antioxidant activity. The achieved outcomes showed that the addition of germinated green buckwheat flour into horsemeat patties lessens the adverse effect of storage and free radical formation and, therefore, can be effectively used to extend the shelf-life of stored patties and to slow down lipid oxidation in meat products.

Effect of GGBF on surface color parameters of uncooked horsemeat patties during storage

Regarding the meat products, the addition of the germinated green buckwheat and its flour changed the structure and consistency of the final product and led to an enhancement of samples from a physicochemical point of view. The effect of the use of buckwheat sprout flour on the surface color of horsemeat patties is given in Table 4. Special consideration was paid to the quantity of the sprouted buckwheat flour added due to its effect on the appearance and properties of the final product. The formulation with the maximum nutritional value and food sufficiency was selected from many recipe alternatives. The color of meat products is a sign of meat quality and freshness, so it is the most, not the least, significant parameter.

Table 4. Color characteristics of raw horsemeat patties formulated with GGB and its flour during storage at 4°C (±1°C).

Parameters	Storage (days)					p-value
	0	7	14	21	23
	L*
Control	72.5 ± 2.81	64.3 ± 3.41	48.2 ± 1.53	48.1 ± 2.35	44.8 ± 1.92	**
Test	70.4 ± 0.42	67.6 ± 0.18	53.5 ± 1.71	59.9 ± 0.41	52.8 ± 1.99	**
	a*
Control	8.4 ± 0.57	9.1 ± 0.38	9.2 ± 1.01	11.5 ± 0.57	10.8 ± 0.48	**
Test	7.4 ± 0.81	9.0 ± 0.61	11.2 ± 1.81	9.3 ± 1.01	12.7 ± 0.71	**
	b*
Control	14.2 ± 0.01	11.7 ± 0.07	12.8 ± 1.51	10.3 ± 0.03	13.2 ± 1.31	*
Test	14.8 ± 0.24	13.5 ± 0.51	15.9 ± 1.62	13.6 ± 0.21	18.4 ± 0.83	**

All values are mean ± standard deviation of triplicates. p-value: ** (p < 0.01), * (p < 0.05)

In the meat production industry, an increase in the brightness of frozen patties is unaccepted because it shows that the non-meat components are in a higher proportion. For the L* value, the control sample had the highest value (p < .05), while the GGBF addition resulted in a lower L* value for the test sample. Also, similar results were given by Ozturk et al.,^[27] who reported that the highest L* values were obtained in control samples. The L* values of both tests decreased slowly during 23 days of storage. Minekata et al.^[14] reported that phenolic concentrations affect the differences in L* values. Similar data of lessening of L* values during storage of beef patties incorporated with Moringa seed flour^[35] were reported. The a* and b* values in the control sample were lower than in GGBF-added tests and showed higher values during storage. These results established that GGBF exhibited a preserving effect on the color of a patty. That could be credited to the antioxidant compounds present in the GGBF since the assimilation of antioxidant molecules stabilizes oxymyoglobin and delays its deterioration.^[36]

Water holding capacity and cooking properties of horsemeat patties with GGBF

WHC is the ability of a food product to substantially hold water against gravity and is one of the foremost characteristics of flour in meat products; therefore, flours are conventionally used to improve freshness and handling characteristics. Table 5 shows the WHC and cooking properties of horsemeat patties incorporated with GGBF. The WHC of horsemeat patties was 77.50 and 79.90 for NGGBF and GGBF, respectively. As protein and fiber content are critical determinants of the ability of the flour to absorb water, the WHC of GGBF could reflect its high protein and fiber content.^[17] Results like ours were reported by Park et al.,^[37] that the inclusion of fermented buckwheat improved the WHC of meat patties.

Table 5. WHC and cooking properties of horsemeat patties.

Parameters	Treatments		p-value
	NGGBF (control)	GGBF (test)
WHC (%)	77.50 ± 1.25	79.90 ± 3.13	*
Cooking yield (%)	87.84 ± 1.43	88.91 ± 0.53	ns
Change of thickness (%)	27.08 ± 8.13	23.25 ± 13.23	*
Change of diameter (%)	11.53 ± 2.25	11.45 ± 1.51	ns
Fat retention (%)	87.33 ± 7.63	95.15 ± 6.83	*
Moisture retention (%)	50.55 ± 1.03	52.25 ± 1.33	*

All values are mean ± standard deviation of triplicates. p-value: * (p < 0.05), ns – not significant

Enhancement of cooking yield in meat and meat products will increase the weight of the product and, therefore, the profit margins as well as gain customers, who like to have juicier and more flavorful food, which is associated with fat and water retention. However, the components used for yield enhancement often provide various benefits. The cooking yield of the samples changed to 87.84 and 88.91 with the addition of NGGBF and GGBF, respectively. The lack of significant change in cooking yield and diameter is probably due to the protein and fiber content in both NGGBF and GGBF, and it could enable a reduction in the amount of meat in the formulation without harming any technological quality. Similar results^[38] showed that adding plant-based proteins to the ground beef patty improved cooking yield by holding more moisture during the cooking process.

Denaturation of meat proteins with the loss of water and fat led to diameter changes.^[39] Thickness changes after cooking were determined as predicted. These findings can be acknowledged as the stabilizing properties of buckwheat flour, which limited the alteration and permitted the retention of the size and shape of the patties during cooking. Shrinkage can be because by the denaturation of protein, evaporation of moisture, and drainage of liquefied fat and juices that affect the textural quality of cooked patties.

Sensory quality and acceptability depend on how much fat can be retained within the body of the patties during cooking and storage. Fat retention of horsemeat patties was 87.33 and 95.15%. These results showed that the incorporation of GGBF led to a slight increase in fat retention compared to NGGBF. Our results are in good agreement with the findings of Park et al.,^[37] where the incorporation of fermented buckwheat flour had a significant effect on fat content. Similarly, Serdaroğlu et al.^[17] and Lopez-Vargaz et al.^[39] found that dried pumpkin pulp and passion fruit albedo showed higher fat retention in beef and pork burgers. The GGBF addition decreased the moisture retention of horsemeat patties compared to NGGBF samples. These findings are in great contrast to the WHC of the patties, where the water-holding abilities increased with GGBF addition. The increase of moisture and fat retention of patties could be accounted for by increases in the water absorption ability of denatured protein, the thermal separation of proteins, and the gelatinization and swelling of starch and fiber.

Texture analysis and sensory evaluation

Table 6 shows the effects of buckwheat and germinated buckwheat on the textural properties of horsemeat patties. The textural parameters of horse patties, such as hardness, springiness, gumminess, and chewiness were slightly decreased with the addition of GGBF. Previous studies found that the incorporation of fermented buckwheat worsened the textural properties of pork patties,^[37] and authors^[40] have reported that up to 3% buckwheat flour addition showed no significant differences in textural parameters. The textural characteristics of the products could demonstrate alterations according to the natural structure and the amount of the non-meat ingredient; and the amount of replaced meat in the formulation. The addition of GGBF contributed to decreasing the hardness of horsemeat patties. These findings may suggest that NGGBF or GGBF addition could be beneficial in the production of horsemeat patties with softer textural properties.

Table 6. Textural properties of horsemeat patties with NGGBF and GGBF.

Parameters	NGGBF	GGBF
Hardness	3.32	2.45
Springiness	6.35	6.16
Cohesiveness	0.54	0.50
Gumminess	1.72	1.26
Chewiness	1.12	0.78

The addition of non-meat ingredients with a specific aroma and color in meat product formulation can alter the sensory properties of final products. Therefore, it is essential to examine the sensory characteristics of the product and add changes to the formulations if necessary. Figure 2 shows the results of a sensory evaluation of horsemeat patties with NGGBF and GGBF.

Figure 2. Sensory properties of horsemeat patties.

One of the most significant factors during the evaluation of processed meat products is the surface color of the patties. Among all sensory parameters, there were no significant differences in color, tenderness, flavor, and overall acceptability. The patties were a little lighter in color due to the color of green buckwheat flour against that of roasted buckwheat flour, which has a darker appearance. The patties with GGBF showed better juiciness and overall acceptability scores. These results may be due to the sprouts added into the formulation. There is limited information about the green buckwheat sprouts used in any food formulation. Xu et al.^[41] reported that the tartary buckwheat sprouts used in steamed bread formulation led to unfavorable reactions in consumer acceptance due to the sprouts’ bitterness and astringency. Sturza et al.^[13] reported similar results. In our example, the panelists did not detect any unpleasant flavor or bitterness: it probably diminished with the mixture of all the ingredients in the formulation.

Conclusion

This research determined that buckwheat sprout flour is a reliable source of polyphenols and free radical scavengers and that it delivers significant antioxidant benefits to horsemeat patties during cold storage (4.0 ± 1.0°C). Moreover, the germinated buckwheat flour addition yielded considerable changes in physicochemical and oxidative characteristics, and in the textural properties of horsemeat patties. Moisture, fat, and pH levels decreased, whereas the ash, carbohydrates, TPC, DPPH, and WHC content increased. Thus, the new horsemeat product “Shygys” has significant nutritional value and acceptable sensory-textural properties. The results obtained in this paper showed the effectiveness of natural antioxidants in sprouted buckwheat in lessening the oxidative change of products made from minced horsemeat. Further research is required to investigate the shelf-life of horsemeat patties with germinated buckwheat stored at −18 to −20°C to fully understand how this additive could influence the microbial count during storage.

Data availability statement

The data that support the findings of this study are available on request from the corresponding author, Miss Zh. Atambayeva.

Disclosure statement

No potential conflict of interest was reported by the author(s).