INTRODUCTIONPeople living in developing countries, such asKazakhstan, need high nutritive products. In thiscontext, meat is considered one of the main sources ofprotein for consumers due to its high nutritive value.However, meat is expensive. Moreover, Kazakhstanhas a specific lack of food-producing animals, and,thus, a low production of meat obtained from domesticand wild ungulates [1]. As a result, the food security ofmeat products there remains unsatisfactory [2]. Hence,Kazakhstan needs to develop a more competitive meatindustry to improve meat production and market [3].A relatively low production of beef in Kazakhstan isbecoming an urgent problem, considering that Muslimsrepresent a large group of Kazakh population, and theydo not eat pork. Poultry meat could also improve proteinintake by Kazakh people. Combining meat with productsof plant origin is highly convenient for several nutritionalpurposes. A recent study by Shakiyeva et al. of thenutritional status of Kazakh people aged over 40 y.o.demonstrated a low fibre intake and excessive levels ofsaturated fatty acids [4]. Most plant proteins have a goodfatty acid profile, which makes them preferable for humanconsumption. In addition, vegetables are an importantsource of fibre. Therefore, the nutritional composition ofvegetables has several benefits for human health.Soya is one of the plant products that could becombined with meat to formulate a new product.Although soya has lower levels of lysine or sulphuramino acids compared with meat, this food product isan important source of protein and fibre [5]. Soya alsopossesses isoflavones, which have been implicated assubstances with important health benefits for more than adecade [6]. A recent research conducted by Ferguson et al.demonstrated the positive effect of moderate consumptionof isoflavones on metabolic response [7]. This propertymakes soya beneficial for consumers suffering fromobesity or insulin resistance [8]. All these aspects justifythe formulation of meat products that combine ordinary orgerminated soya flour with poultry meat.The introduction of innovative flour-based functionalfoods into the market demonstrated a positive economiceffect [9]. However, the high initial bacteria load in rawsoya, raw germinated soya, and poultry meat is one ofthe main problems associated with this type of product.Moreover, meat is an excellent nutritional source forseveral types of bacteria, even taking into considerationCopyright © 2019, Sholpan et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.Foods and Raw Materials, 2019, vol. 7, no. 2E-ISSN 2310-9599ISSN 2308-4057Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-396-402Open Access Available online at http:jfrm.ruRaw poultry meatballs with soya flour: Shelf life and nutritional valueAmanova Sholpan1, Alexandre Lamas2 , Alberto Cepeda2 , Carlos M. Franco2,*1 Almaty Technological University, Almaty, Republic of Kazakhstan2 Universidade de Santiago de Compostela, Lugo, Spain* e-mail: carlos.franco@usc.esReceived July 19, 2019; Accepted in revised form September 17, 2019; Published October 21, 2019Abstract: Poultry meat is a valuable source of protein for human consumption. It plays an important role in countries with poorungulate meat production, including the Republic of Kazakhstan. The intake of fibre by the Kazakh population also remains low,while the intake of saturated fatty acids is excessive. Therefore, it is recommended to combine meat with plant products, e.g. soyaflour. In the present research, we developed and evaluated a new meatball product containing different amounts of soya flour. Themeatballs proved to be a semi-finished high-protein product. They also demonstrated a good fatty acid and mineral profile. Theproduct with 30% of soya flour showed the best results: 27% of protein, low content of saturated fatty acid, and shelf life of 48 h. Toextend the shelf life of the meatballs under refrigerator conditions, new disinfection methods should be developed.Keywords: Soya flour, chicken, meatballs, nutritional value, predictive microbiology, shelf lifePlease cite this article in press as: Sholpan A, Lamas A, Cepeda A, Franco CM. Raw poultry meatballs fortified with soya flour asa highly nutritive product for Kazakh consumers: shelf life and nutritional values. Foods and Raw Materials. 2019;7(2):396–402.DOI: http://doi.org/10.21603/2308-4057-2019-2-396-402.397Sholpan A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 396–402that this type of product requires thermal treatment.Therefore, producers of this type of food should be carefulwhen defining the shelf life and storage conditions.The present paper introduces meatballs containingminced poultry meat with different percentages ofgerminated and non-germinated soya flour. Direct andindirect methods, e.g. predictive microbiology, wereemployed to determine the nutritional composition,minerals, heavy metals, and the shelf life of the newproduct.STUDY OBJECTS AND METHODSFormulation and elaboration. The meatballs weremade of chicken breast. The minced chicken meat wascombined with different concentrations and types ofgerminated and non-germinated soya flour to producefour different meatball samples. Sample A contained70% of minced chicken and 30% of dry germinated soyaflour. Sample B included 85% of minced chicken and15% of dry germinated soya flour. Sample C contained70% of minced chicken and 30% of dry non-germinatedsoya flour. Sample D consisted of 85% of minced chickenand 15% of dry non-germinated soya flour. The meatballswere stored at 4–6°C during the research process.Microbiological analysis. The microbiologicalanalysis was performed on days 0, 3, and 7. Thesamples were tested for Salmonella spp. and Listeriamonocytogenes and proved to contain neither. 25 g ofmeatballs were homogenised with 225 mL of bufferedpeptone water (Merck, Germany) in a stomacher(MIX2, AES-Laboratory, France) for 2 min. Thetotal viable counts were evaluated in plate count agar(Liofilchem, Teramo, Italy) incubated at 32ºC for 72 h.Enterobacteriaceae were counted in violet red bileglucose agar (Liofilchem) incubated at 32ºC for 24 h.Coliforms were detected in violet red bile lactose agar.Escherichia coli were detected in Fluorocult® (Merck)incubated at 42ºC for 24 h. The presence of presumptiveStaphylococcus aureus was evaluated in Baird–Parkeragar (bioMérieux, Marcy l´Étoile, France) incubated at37ºC for 48 h. The presence of Salmonella was detectedaccording to ISO 6579-1:2017 [10]. The meatballhomogenate prepared as described above was incubatedat 37ºC for 24 h. A 100-μL aliquot of the incubatedpeptone water was transferred to 10 mL of Rappaport–Vassiliadis (RV) enrichment broth and incubated at42ºC for 24 h. Next, one RV broth loopful was streakedon xylose-lysine-deoxycholate agar (Oxoid) andSM-ID2 (bioMérieux) and incubated at 37ºC for 24 h.Listeria monocytogenes was determined according toISO 11290-1:2018 [11]. Twenty five grams of meatballswere incubated in half-strength Fraser broth (Oxoid) at30ºC for 24 h. Then, 100 μL was transferred to a tubecontaining 10 mL of Fraser broth and incubated at 37ºCfor 48 h. Finally, the half- and full-strength Fraser brothswere plated out on Aloa® agar (bioMérieux), and theplates were incubated at 37ºC for 48 h. All analyses wereperformed in duplicate.Predictive microbiology. The data obtained for themicrobiological analysis were compared with the dataand scenarios obtained from ComBase, www.combase.cc (University of Tasmania, Tasmania, Australia; andthe USDA Agricultural Research Service, Beltsville,MA, USA), which is a free on-line modelling databasefor predictive microbiology. The parameters used werethose obtained from the initial analysis of the meatballs.Several conditions for bacterial growth were testedto assure the results obtained for the shelf life of theproduct.pH measurement. The pH level was measured usinga Crison PH 25+ pH meter with a penetration electrode(Crison Instruments, Barcelona, Spain) by introducingthe electrode into the meatballs. Determinations foreach treated meatball were performed in triplicate everythree days.Nutritional analysis. All analyses for the proximatecomposition were performed using standard AOACmethods [12]. The moisture content was determined bydrying samples in a laboratory drying oven (Selecta,Barcelona, Spain) until the weight became constant. Thetotal protein was determined by the Kjeldahl method.A factor of 6.25 was used to convert total nitrogen intocrude protein. The samples were digested using a Kjeltec1007 digester (Tecator, Höganäs, Sweden) and distilledusing a Kjeltec 1026 distilling unit (Tecator). The lipidcontent was assayed by extraction with diethyl ether/petroleum benzene (1/1, v/v) in a Soxhlet system (SoxtecHT 1043, Tecator). The ash content was assessed byincineration in a muffle furnace (Utena, Lithuania). Thecarbohydrate quantity and energy content were obtainedby calculations.Minerals and heavy metals. Minerals and heavymetals were analysed by the method of inductivelycoupled plasma-optical emission spectrometry(ICP-OES). One gram of sample and 4 mL of 69%HNO3 (Hiperpur, Panreac, Spain) were homogenisedin glass tubes and incubated at room temperature for1 h. Afterwards, 1.5 mL of 33% (w/v) H2O2 (Panreac)was added, and the mixture was heated first at 120ºCfor 10 min in a heater block (Selecta) and then at 130ºCfor 3 h. After the samples were cooled down to roomtemperature, Milli-Q water was added until the volumereached 25 mL. The samples were analysed in anOptima 4300 DV ICP-OES (PerkinElmer, MA, USA)under the following conditions: plasma flow, 15 L/min;auxiliary flow, 0.2 L/min; nebuliser flow, 0.8 L/min;sample flow, 1.5 mL/min.Urease activity. The urease test was conducted asfollows: 10 mL of a buffered urea solution (pH = 7.0)was added to 0.2 g of finely ground soya (test sample),and 10 mL of a phosphate-buffered solution wasadded to 0.2 g of the same sample (blank sample).The two solutions were incubated at 30ºC for 30 minunder stirring. In the presence of significant ureaseactivity, the pH of the test solution increased due to the398Sholpan A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 396–402ammonia released from the urea. Immediately after theincubation, the pH of the solutions was determined, andthe difference between the pH of the test and the blanksamples was calculated as the urease activity index. ThepH was measured as described in section 2.4.RESULTS AND DISCUSSIONTable 1 shows the microbiological analysis results ofthe four samples.The total bacterial count, as well as coliform andenterobacteria counts were higher for the samplewith germinated soya flour compared with that withnon-germinated soya flour. The values reached onelogarithmic cycle or above. This result was expected,since germinated soya contains more sugars thanoligosaccharides, which should be a good advantage forbacterial growth [13]. However, the E. coli counts werevery similar in all the samples.L. monocytogenes and Salmonella were notisolated from any of the analysed samples. Variationsin Salmonella prevalence depend on the origin of thepoultry meat, as shown in [14]. Despite the importanceof Salmonella tests for poultry production, the incidenceof this foodborne pathogen has decreased in recent years[15]. In the case of L. monocytogenes, the past centurysaw a substantial improvement in quality regardingthe prevalence of this microorganism in food [16]. Forinstance, the prevalence of L. monocytogenes in poultrybreast was reported to reach 20% in 1990s. However, ithas dropped to 8.6% in the last few years [17].According to the ComBase results obtained forSalmonella-positive poultry samples, a value of3.68 log CFU/g could be reached after one week underthe following conditions of storage: temperature, 7ºC;pH, 6.4; a physical state for bacteria, 1; no lag phase.Comparatively higher values of L. monocytogenescould be reached, even at temperatures below 7ºC,for the same storage time. The total aerobic countwas a good indicator of the shelf life of the product,and values above 7 log CFU/g indicated a markedalteration in the meatballs. The product needs to bestored at refrigeration values. Thus, Pseudomonads orBrochothrix thermosphacta can be selected in ComBaseto predict the storage stability of the meatballs, as thesebacteria are frequently related to meat spoilage [18, 19].For Pseudomonads and B. thermosphacta, valuesabove 7.5 log CFU/g could be achieved at 48 h of storageunder the following conditions: temperature, 5ºC; pH,6.4; water activity, 0.99; initial value, 5 log CFU/g;physical state for bacteria, 1; no lag phase. However,the meatballs developed in this study showed valueshigher than 7.5 log CFU/g (total aerobic count) afterjust one week of storage. The fact that a physical stateof 1 implies no lag phase presupposes an extreme casethat could rarely occur in real situations. In any case, asemi-manufactured product, such as the meatballs understudy, could only have a maximum shelf life of 48 h atrefrigeration temperature. The obtained data and thefact that the meatballs contained only raw ingredientsproved that an adequate shelf life could be achievedby packaging or disinfection methods, e.g. ionisingradiation, or refrigeration [20].The values obtained for the urease activity confirmedthe absence of thermal treatment in soya flour. ThepH value ranged from 7.14 for the product with only15% of germinated soya flour to 8.30 for the productwith 30% of germinated soya flour. In the controlsamples and the samples with the cooked soya flour,the values were always ≤ 7 due to the absence of ureasein the treated product. For the products with 15 and30% of germinated soya flour, pH was 6.63 and 6.82,respectively According to Craven and Mercuri [21],several commercial texturised soya proteins were usedin meat products with no increase in bacterial countsrelative to the control samples. In the present study, theless processed soya flour caused higher bacterial counts.Table 1 Microbial counts and pH on day 0, 3, and 7 in meatball samples with soya flourSamples (mincedmeat/soya proportion)Day pH TAC* EnterobacteriaceaeColiforms EscherichiacoliStaphylococcusaureusListeria monocytogenesSalmonella70/30 germinated soya 0 6.44 8.8×106 6.2×105 1.8×104 2.0×102 < 50 nd nd3 6.89 4.3×107 3.2×106 6.4×105 2.6×102 < 50 nd nd7 7.12 9.1×108 6.8×106 1.2×106 3.3×103 < 50 nd nd85/15 germinated soya 0 6.33 5.0×106 2.4×105 1.0×104 3.1×102 < 50 nd nd3 6.78 6.3×107 9.2×105 2.4×105 8.6×102 < 50 nd nd7 6.99 9.8×108 3.8×106 5.9×105 3.6×103 < 50 nd nd70/30 non-germinated soya 0 6.31 3.5×105 4.5×104 2.0×103 1.2×102 < 50 nd nd3 6.84 4.8×106 2.2×105 2.4×105 4.5×102 < 50 nd nd7 6.92 4.2×107 2.8×106 5.9×105 9.2×102 < 50 nd nd85/15 non-germinated soya 0 6.39 1.7×106 6.2×105 9.0×103 1.5×102 < 50 nd nd3 6.90 5.2×107 1.5×106 3.3×104 1.8×102 < 50 nd nd7 7.21 2.2×108 9.3×106 8.5×105 4.1×102 < 50 nd nd*TAC: total aerobic counnd: not detected399Sholpan A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 396–402As for nutritional properties, the idea of using soyaprotein is more than 30 years old [21]. Chicken breastwas found to achieve a maximum protein content of34.5%, although values of 24% are most frequentlyreported [22, 23]. For the products developed andstudied in the present research, similar values to thosedescribed by Lonergan et al. [23] were obtained in themeatballs prepared with 15% of soya flour. However, animportant increase in protein content was observed forthe meatballs with 30% of germinated soya flour. Theirprotein level was ≥ 27% (Table 2).The primary objective of the present study was toobtain a high-protein product. The research proved thatcould only be achieved by adding 30% of soya flour tothe meatballs. Of the two samples with 30% of soyaflour, the meatballs with non-geminated soya flourshowed better results, with 2% more protein than in thesamples that contained germinated soya flour.The meatballs with 30% of soya flour proved tohave nutritional advantages. In addition, combination ofprotein and fibre can promote satiety [24]. The productswith 30% of soya flour had a higher dry weight valuesthan those with 15% of soya flour. This fact couldtrigger a higher water intake and, hence, an increase insatiety. Satiety is an important aspect to consider bothfor diabetes and/or dietetic treatment of obesity. Theeffect of high protein intake on satiety is so strong thata remission of pre-diabetes to normal glucose tolerancewas observed in patients fed with a 30% dietetic proteinfor 6 months [25]. As revealed above, isoflavones in soyacould also help to improve insulin tolerance [7, 8].Serdaroglu et al. and Ikhlas et al. studied the qualityof low-fat beef meatballs with 10% of various legumes,excluding soya flour [26, 27]. They reported lower proteinvalues (≤ 24%) than those obtained for the productsdeveloped in the present study. Increased amounts oflegume flour were suggested to be used as extenders formeatballs [26]. The meatball samples used in the presentresearch showed no differences in total, saturated, andunsaturated fat (Table 2). Their values were always≤ 4% and sometimes even ≤ 3%. Judging from these fatcontents, the proposed meatballs had lower energy valueand fat content than beef, pork, or even some turkey partsor duck meat [22]. Likewise, the low amount of saturatedfat together with the high polyunsaturated fat content canimprove traditional Kazakh diet.As stated in [28], saturated fats should provide about7% of dietary energy. The content of saturated fats in theTable 2 Nutritional composition of meatball samples with soya flourParameter Samples (minced meat/soya proportion)70/30 germinated soya 85/15 germinated soya 70/30 non-germinated soya 85/15 non-germinated soyaDry weight 43.45 33.40 45.31 35.03Protein 27.18 24.37 29.49 25.69Fat 2.85 2.52 2.17 3.20Saturated fat 0.73 0.65 0.50 0.82Monounsaturated fat 0.57 0.54 0.44 0.66Polyunsaturated fat 1.55 1.33 1.23 1.72Ash 2.23 1.79 2.53 1.66Carbohydrate 11.19 4.79 11.12 4.48Energy, kcal/100 g 179.13 139.04 181.97 149.48Sodium, mg/100 g 40.38 48.97 45.18 53.57Table 3 Minerals and heavy metals in meatballs with soya flourMinerals and heavymetals, mg/kgSamples (minced meat/soya proportion)70/30 germinated soya 85/15 germinated soya 70/30 non-germinated soya 85/15 non-germinated soyaMg 656.22 393.57 476.01 421.97P 1872.63 1520.86 1636.46 1629.99K 5652.08 3930.54 4987.42 4360.88Ca 802.88 386.48 476.07 318.47Fe 16.14 9.20 14.27 10.97Ni 0.50 0.21 0.35 0.6Cu 3.58 1.33 2.51 1.51Zn 11.09 8.02 9.36 8.66As 0.0065 0.0055 0.0056 0.0033Se 0.0956 0.1097 0.1067 0.1255Cd 0.0064 0.0033 0.0047 0.0031Hg 0.0104 0.0063 0.0055 0.0046Pb 0.0065 0.0034 0.0032 0.0013400Sholpan A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 396–402meatballs was ≤ 1%. Therefore, 200 g of the meatballscontained 60 g of protein and about 2 g of saturated fats,i.e. 240 and 18 kcal, respectively. It complies with thegeneral recommendations for saturated fat intake.As for minerals and heavy metals, the four meatballsamples were tested for a total of 13 elements (Table 3).Remarkably, the meatballs with 70% and 30% ofgerminated soya flour demonstrated higher contents ofMg, P, K, Ca, Fe, and Zn and a double or more of Cd,Hg, and Pb than the other samples. These results wereprobably due to the higher mineral content found inthe soya flour, as the content increased with soya flourconcentration in the produced meatballs. Therefore,plant food products can be expected to have higheramounts of minerals than animal food products.The study conducted by a Chinese research teamshowed high levels of As, Cu, and Zn in poultry tissues,which were mainly attributed to feed supplements [29].The Chinese study proved that the amounts of As foundin inorganic poultry meat in Lianzhou and Guangzhoupose a significant public health risk, considering thehigh level of bladder or lung cancer in these cities. Inthe products designed in this study, the As level wasan order of magnitude lower than that obtained by Huet al. [29]. The levels of Cd and Pb in the present studywere also lower. However, we detected higher levels ofCu and Ni. In any case, the soya flour used in this studywas poorly processed and did not undergo any thermaltreatment, as verified by the urease test.Soya has important anti-nutritive agents, so thisproduct has to be treated to avoid the effect of thesecompounds. These anti-nutritive factors are phytic acid,phytates, and protease trypsin inhibitors. The presenceof natural phytates, for instance, significantly increasesthe calcium requirements. In soybeans, the phytic acidcontent is 1.00–1.47% of dry weight, which meansmore than 50% of phosphorous [30]. The treatments toeliminate trypsin inhibitors from soybean flour wererecently reviewed by Vagadia et al. [31]. The cookingof soya flour in an alkaline system at 90ºC for 15 min issufficient to inactivate the protease trypsin inhibitors.CONCLUSIONThe present research introduced, developed, anddescribed a new poultry meat product: meatballsformulated with germinated or non-germinated soyaflour. The use of 30% of soya flour resulted in a semifinishedhigh-protein product. The soya flour used in theformulation produced a number of other positive effects,e.g. low and well-balanced fat content and increasedamounts of fibre and isoflavones. The increase inmineral content could depend on the specific plant originof the soya flour, and additional treatments are necessaryto avoid the negative effect of anti-nutritive compounds.Direct microbiological analyses and predictivemicrobiology showed that the mixture of minced poultrymeat and soya flour produced a product with a shelf lifeof 48 h. In order to extend the shelf life of the product,specific packaging procedures or disinfection techniquesshould be applied.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest related to this article.