INTRODUCTIONCurrent global trends in food production are aimedat designing healthy foods to improve public health andprevent diseases caused by unbalanced nutrition. In therecent years, the quality and safety of food products hasbeen a strategic priority in Russia. New laws have beenpassed to regulate and encourage the development andproduction of a wide range of healthy foods, includingfunctional products.Diseases of the musculoskeletal system areamong the most common in modern clinical practice,especially osteoporosis. According to the WorldHealth Organization, almost 200 million people sufferfrom osteoporosis worldwide, with over 9 millionfractures occurring every year. Women aged 55+are especially vulnerable to this pathology, which ispresumably associated with estrogen deficiency in thepostmenopausal period.Postmenopausal osteoporosis is caused byaccelerated bone resorption and systemic calciumimbalance. Osteoporosis caused by hypoestrogenismis commonly treated with drugs that prevent boneresorption or stimulate the formation of bone tissue.These drugs are mainly based on female sex hormonesor selective estrogen receptor modulators. However,hormone therapy in postmenopausal women canbe a risk factor for stroke, myocardial infarction,thromboembolism, and breast cancer. Moreover, thesedrugs can cause serious side effects, such as atrialfibrillation, atypical subcutaneous fracture, delayedfracture healing, hypersensitivity reactions, hot flashes,leg cramps, gastrointestinal disorders, etc. Anothercause of osteoporosis is deficiency states due toinsufficient intake of calcium, magnesium, protein, andvitamin D [1–8].All these factors determine a need for new waysof osteoporosis prevention and treatment, namely for355Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–363alimentary correction with functional foods. Such foodsnot only meet the intake of essential nutrients, but alsobenefit certain bodily functions and prevent the negativeeffects of lifestyle and environmental factors. Our diet –as a whole and its individual components – influencesdifferent physiological processes in our body. Therefore,food formulators should introduce physiologicallyactive ingredients with corrective properties, as wellas use technology that preserves the nutritional andbiological value of raw materials and components duringprocessing and cooking.Formulation of functional meat products is oneof the current trends in modern meat industry. Inparticular, low-value meat-and-bone material can beused to obtain protein hydrolysates and bone mineralcomponents [9–12].Protein hydrolysates are commonly used as analternative protein source in commercial products.They consist of a mixture of proteins and peptidesresulting from the hydrolysis of intact proteins. Duringhydrolysis, peptide bonds of intact proteins get broken,which leads to a range of peptides of different sizes.Protein hydrolysates are used in various productsdepending on their properties [13, 14]. Numerousstudies show that protein hydrolysates can be used indiets due to their high nutritional and therapeutic value– low immunological reactivity, bioactive peptides, andantioxidant activity. Protein hydrolysates are widelyused in the diet for people who cannot digest wholeprotein. Protein hydrolysis can be carried out usingenzymes, acids, or alkalis, but enzymatic hydrolysisis preferable for food purposes since it can producehydrolysates with a well-defined peptide profile [15].Collagen-containing products of meat processingare the main source of collagen with a unique aminoacid composition. Collagen can be transformed intoactive peptides and amino acids to be used as functionalingredients in food formulations. It is a protein that ispresent in large quantities in the connective tissue ofanimal materials. Connective tissue is part of cartilage,tendons, subcutaneous tissue, bone, intercellularsubstance of muscles, parenchymal organs, and vascularwalls. It accounts for about 50% of the animal’s bodyweight. Connective tissue contains proteoglycans,whose polysaccharide group includes glucosamine orgalactosamine. One of its main functions is that it takespart in the formation of organs and their restoration.Enzymatic hydrolysis increases the bioavailabilityof collagen and glycosaminoglycans for the body toassimilate. It produces peptides and amino acids that areabsorbed into the bloodstream and then enter the cells ofthe connective tissue matrix [16].The content of protein fractions and their amino acidcomposition in hydrolysates can be regulated by modesof hydrolysis, type of enzyme, processing method,temperature, and other factors.Pork legs and ears are a valuable source of collagenhydrolysates. They contain 23.5 and 21.0% of protein,respectively, with 15.3 and 12.6% in the connectivetissue, respectively. In our previous work, wesubstantiated hydrolysis parameters for the productionof active peptides and free amino acids (10–15%) [17].In particular, we described a method for obtaininghydrolysate from pork legs and ears using enzymecontainingpancreas homogenate (15% of the rawmaterials.) at T = 50 ± 2°C, τ = 6 h, and further freezedryingat –40°C.Thus, functional foods play a special role in theprevention and nutritional correction of osteoporosis,especially products based on hydrolyzed connectivetissue proteins. They contain large amounts of collagenpeptides and amino acids that stimulate the synthesisof physiological collagen and other substances creatingcartilage and bone matrix. In addition to collagenpeptides, the diet should meet the intake of calcium,magnesium, copper, zinc, as well as vitamins D, A, E, C,and group B.In this regard, we aimed to formulate a functionalproduct based on collagen fermentolysate forosteoporosis prevention and to confirm the identifiedproperties in animal experiments. Our objectiveswere to substantiate the formulation in terms of itscomposition and component ratios, evaluate its sensory,microbiological, and toxicological indicators, as well asassess its restorative effect on bone metabolism impairedby oophorectomy in experiments on rats.STUDY OBJECTS AND METHODSOur study objects were dried collagen fermentolysateand a functional product based on it.Collagen fermentolysate was obtained from porkby-products (ears and legs, 1:1) using raw pancreashomogenate as an enzyme-containing material. Theresulting hydrolysate was dried under vacuum in anAlpha 1-2 LD freeze-dryer (Germany) at –40°C. Then,it was crushed to a particle size < 0.2 mm. The resultinghydrolysate was a homogeneous fine powder of lightbeige color, readily soluble in water.The molecular weight distribution of proteinfractions in the collagen fermentolysate was studiedby electrophoresis in a 10% polyacrylamide gel withsodium dodecisulfate (SDS) according to Laemmli. Theamino acid composition was determined on an Agilent1260 Infinity LC liquid chromatograph in line with StateStandard 34132-2017. The hydroxyproline content wasmeasured in line with State Standard 23041-2015.Protein content in the product was determined bythe Kjeldahl method according to State Standard 25011-2017. Mass fractions of vitamin D3 and calcium weremeasured according to State Standard 32307-2013 andState Standard R 55573-2013, respectively (the latter byatomic absorption). The method to determine vitamin Cinvolved the vitamin’s extraction (by sequential acid andenzymatic hydrolysis), precipitation of proteins, and highperformance liquid chromatography in the ultraviolet(UV) region at a given wavelength. The resulting peakin the chromatogram was compared with the peak of astandard with a known concentration.356Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–363The method to determine vitamin E was based onalkaline hydrolysis of the sample and extraction withdiethyl ether. The obtained extract was analyzed by highperformance liquid chromatography in the ultraviolet(UV) region at a given wavelength. The resulting peakin the chromatogram was compared with the peakof a standard vitamin solution with a known massconcentration.Microbiological indicators were determined usingthe following standards:– State Standard 10444.15-94 for the quantityof mesophilic aerobic and facultative anaerobicmicroorganisms (QMAFAnM);– State Standard 31747-2012 for coliform bacteria;– State Standard 31659-2012 for salmonella bacteria; and– State Standard 10444.12-2013 for mold.Toxic elements were determined according to thefollowing standards:– State Standard 26927-86 for mercury;– State Standard 26930-86 for arsenic;– State Standard 26932-86 for lead; and– State Standard 26933-86 for cadmium.Biological experiments were carried out on femaleWistar rats (n = 42) weighing 340 ± 20 g. The animalswere kept and studied in a vivarium in strict accordancewith State Standard 33216-2014.After quarantine (7 days), the rats were randomlydivided into two groups: 1) intact rats (n = 10), whowere fed on a standard diet throughout the experiment,and 2) rats exposed to experimental osteoporosismodeling (n = 32).The standard vivarium diet contained 12% caseinproteins, 72% soluble carbohydrates, 11.5% saturatedand polyunsaturated fatty acids, 1.0% fat-solublevitamins, 0.1% water-soluble vitamins, and 4.0%minerals [18].The osteoporosis modeling involved completeoophorectomy under general anesthesia (Zoletil 100,Virbac S.A., France; Xila, Interchemie, Netherlands).After 14 days from the surgery, the ovariectomizedfemale rats were divided into three groups:1) control animals (control), which daily receivedintragastrically administered distilled water (0.5 ml/head) for 28 days (n = 10);2) experimental animals (experiment 1), which dailyreceived an intragastrically administered glucosaminechondroitinsolution in a dose of 0.014 g per 1 kg of liveweight (Pharmacor Production, Russia) (0.5 ml/head) for28 days (n = 11); and3) experimental animals (experiment 2), which dailyreceived an intragastrically administered functionalproduct based on collagen fermentolysate and dissolvedin water (12 g/100 mL) in an amount of 0.5 g per 1 kg oflive weight (0.5 mL/head) for 28 days (n = 11).The rats were kept in IV S cages (Tecniplast, Italy),5 animals each, under standard vivarium conditions:temperature 20 ± 3°C, humidity 48 ± 2%, day/nightlighting (from 6.00 to 18.00), as well as free access towater and feed [18].Before the study and after administering thefunctional product, the animals were weighed every4 days on a laboratory electronic balance (AdventurerPro AV2101, USA). On the 42nd day of the experiment,the animals were euthanized in a chamber (VetTech,UK), with blood samples extracted from the heart.The experiments were conducted in compliancewith Order No. 267 of the Russian Ministry of Healthof June 19, 2003 “On the rules of laboratory practice”and European Community Directive 86/609EEC. Thestudy was approved by the bioethical commission of theV. M. Gorbatov Federal Research Center of FoodSystems (protocol No. 01/2019 of May 09, 2019) [18].Following autopsy, all the animals underwent athorough examination of their body surface, as well asintracranial, thoracic, and abdominal cavities and theircontents. Their internal organs (liver, kidneys, spleen,adrenal glands, thymus, and heart) were separated andwet-weighed immediately after dissection. Femurs weresampled to determine mass fractions of phosphorus(State Standard 32009-2013), magnesium (State Standard33424-2015), and calcium (State Standard R 55573-2013).The blood cytometric assay involved countsof lymphocytes (LYM), granulocytes (GRA), andFigure 1 Molecular weight distribution of protein-peptide fractions in collagen fermentolysate051015202530354045>400-600 230-400 170-230 100-170 40-100 20-40 10-20Total protein, %Molecular weight, kDa6810121416acids, g/100 g protein357Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–363monocytes (MON) according to cell size and granularityon a Guava Easy Cyte flow cytometer (Merck Millipore,Germany). The content of leukocytes was determined asa sum of lymphocytes, granulocytes, and lymphocytes.Relative contents of lymphocytes, granulocytes, andmonocytes were calculated using the formulas: LYM/WBC×100%, GRA/WBC×100%, MON/WBC×100%.Biochemical parameters of blood serum weredetermined on an automatic BioChem FC-360 analyzer(HTI, USA) using a set of reagents (HighTechnology,USA) [18]. Biochemical analysis measured total protein,albumin, bilirubin (total and direct), urea, creatinine,triglycerides, aspartate aminotransferase (ASAT),alanine aminotransferase (ALAT), alkaline phosphatase(ALP), gamma-glutamyltransferase (GGT), lactatedehydrogenase (LDH), calcium, cholesterol, glucose,phosphorus, and magnesium.Osteocalcin in blood serum was quantified byenzyme-linked immunosorbent assay (ELISA) usinga set of rat-specific reagents on an Immunochem 2100analyzer (HTI, USA).Statistical analysis was performed in STATISTICA 10.Statistical significance was determined by the Kruskal-Wallace H-test (P ≤ 0.05).RESULTS AND DISCUSSIONFirst, we studied the physicochemical parametersof collagen fermentolysate. The molecular weightdistribution of its fractions is shown in Fig. 1.As can be seen in Fig. 1, about 41% of fractionsweighed from 10 to 20 kDa. Peptides with sucha molecular weight should be used as the basisof a functional beverage, since they ensure highbioavailability and good taste characteristics.The amino acid composition of collagenfermentolysate is shown in Fig. 2.According Fig. 2, collagen fermentolysate containedrelatively high contents of glutamic acid (14.8%),aspartic acid (10.8%), glycine (7.3%), alanine (6.9%),and proline (4.9%). These amino acids are known tostimulate cartilage and bone cells and restore jointtissues. Alanine is the main component of connectivetissue, while proline and lysine are precursors ofhydroxylysine and hydroxyproline, which are used bythe body to form collagen, tendons, and heart muscle.We also found high contents of leucine and threonine.These essential amino acids are important for thebiosynthesis of glycine and serine, which are responsiblefor the production of collagen, elastin, and muscle tissue.Based on collagen fermentolysate, our functionalproduct with an antiosteoporosis effect also containeddietary fiber, bioactive substances, as well as macro- andmicroelements.The formulation was in line with the biomedicalrequirements for the quality, composition, and safety offunctional products with corrective properties. To havea real physiological effect, the product should contain atleast 50% of collagen fermentolysate. However, it shouldalso have good consumer appeal. To neutralize the flavorof fermentolysate, pumpkin powder was used as dietaryfiber. It has a pleasant taste and, at the same time,contains various carbohydrate components, includingpectins, cellulose, fiber, calcium, magnesium, iron, Bvitamins, vitamin PP, beta-carotene, and vitamin C.Our formulation was primarily aimed at normalizingmetabolic processes and preventing diseases of themusculoskeletal system. The component quantities metthe physiological needs of adult humans.Increased calcium intake is an integral part ofosteoporosis prevention and treatment. To assimilatecalcium, we added vitamin D3, as well as vitamins Eand C with antioxidant properties. Products based oncollagen hydrolysates in combination with vitamin Care more effective in stimulating collagen fibrils andproteoglycans in the cartilage matrix, thus improvingjoint mobility [19]. Oxidative stress is an importantfactor of aging that also contributes to osteoporosis. Itinduces bone resorption due to superoxide production byosteoclasts, which leads to bone degradation [5, 20, 21].The dietary fiber included in the formulation isa prebiotic that ensures normal functioning of thegastrointestinal tract and has a beneficial effect on lipidand carbohydrate metabolism. In addition, indigestibleoligosaccharides increase the absorption of variousminerals, contributing to bone mineralization [22].Figure 2 Amino acid contents in collagen fermentolysate051015202530354045>400-600 230-400 170-230 100-170 40-100 20-40 10-20Total protein, %Molecular weight, kDa0246810121416Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-Cys Val Met Phe Ile Leu Lys OPro ProAmino acids, g/100 g protein0.81.21.62.0elements, mg/kg358Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–363Of great importance are chondroprotectors –glucosamine sulfate and chondroitin sulfate. They have apositive effect on metabolic processes in cartilage tissue,slowing down degenerative changes in joints and thespine.Our choice of ingredients was determined by twomain objectives. Firstly, we aimed to formulate anefficient functional product with a high nutritionalvalue. Secondly, we wanted this product to have goodsensory characteristics. Both objectives could beachieved with pumpkin powder of the Gribovskayavariety. The pumpkin was peeled, cut into 5–8 mmpieces, blanched for 3–5 min, and placed on racks for10–15 min to remove water. The pieces were then driedin a convection drying chamber in two stages – first,at 90 ± 5°С to a moisture content of 40–42% and then,at 60 ± 5°C to a moisture content of 3.0–5.0%. The driedpumpkin was crushed to a particle size of 0.2 mm. Theresulting powder had a sweetish taste and yellowishcolor.To determine an optimal ratio between collagenfermentolysate and dried pumpkin, in both functionaland sensory terms, we carried out a sensory experiment.The panelists preferred the taste characteristics of a80:20 ratio between protein hydrolysate and pumpkinpowder. A higher content of hydrolysate gave theproduct a pronounced bitter taste, which was consideredunacceptable.The amounts of functional ingredients had to meetthe standard physiological needs without spoilingthe consumer appeal. These ingredients includedcalcium lactate, glucosamine sulfate, chondroitinsulfate, ascorbic acid (vitamin C), tocopherol acetate(vitamin E), and cholecalciferol (vitamin D3).The formulated product is a dry powder forpreparing a functional drink (Table 1).Pre-mixtures based on the compatibility and finenessof ingredients were introduced into a drum-type mixerin two stages to ensure uniformity. The first premixtureincluded fine ingredients in smaller quantities(vitamins D3, E, and C, chondroitin sulfate, andglucosamine sulfate). At the second stage, they werecombined with the remaining components (proteinhydrolysate, calcium lactate, dried pumpkin, and dietaryfiber). A drink can be prepared by mixing 12 g of theconcentrate with 100 mL of water. Three servings perday are needed to provide a good preventative effect.Next, we studied the sensory, physicochemical,microbiological, and toxicological indicators ofthe developed product. The nutritional value of thepowdered product is shown in Table 2.When formulating a functional product based onhydrolysates, it is important to crease a characteristicsensory profile. The sensory indicators of our functionalproduct, both in powdered and ready-to-use form, arepresented in Table 3.The microbiological properties and contents of toxicsubstances (lead, arsenic, cadmium, and mercury) in theTable 1 Product formulationComponents Mass fraction, %Collagen fermentolysate 56.00Dried pumpkin 20.00Inulin 11.00Calcium lactate 7.00Glucosamine sulfate 3.00Chondroitin sulfate 1.00Ascorbic acid (vitamin C) 0.41Tocopherol acetate (vitamin E) 0.09Cholecalciferol (Vitamin D3) 0.13×10–4Table 2 Nutritional value of the powdered productComponent Contentper 100 gpowderContent perserving (% ofdaily intake)Proteins, g 50.0 6.0Carbohydrates, g 14.0 2.0Dietary fiber, g 12.0 1.4 (7%)Ascorbic acid, mg 410.0 49.0 (80%)Tocopherol, mg 90.0 10.8 (108%)Cholecalciferol, μg 13.0 1.6 (31%)Calcium, mg 1100.0 140.0 (15%)Glucosamine sulfate, mg 3000.0 360.0 (51%)Chondroitin sulfate, mg 1000.0 120.0 (20%)Table 3 Sensory characteristics of the functional productIndicator Product characteristicsPowder DrinkAppearance Fine, light yellow powder consistingof single agglomerated particlesTransparent light yellow liquid without sedimentConsistency Loose, agglomerated particles disintegrateunder light mechanical impactLiquid, homogeneous, without settlingColor Light yellow Light yellow, intense, with no glossAroma Mild, with a pumpkin note Mild, with a light pumpkin noteTaste – Pleasant, slightly sweet, with a pumpkinflavor, and slight sourness359Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–363functional product were analyzed against the TechnicalRegulations of the Customs Union 021/2011 “On foodsafety” (Table 4 and Fig. 3).We found that the concentrations of lead and arsenicwere significantly below the permissible values, andthe contents of cadmium and mercury were within thenorms established by TR CU 021/2011. This means thatour functional product met the safety requirements ofTR CU 021/2011.Thus, we developed a powdered functionalproduct based on collagen fermentolysate to preventosteoporosis. Mixed with water, the drink can be usedas an additional source of connective tissue protein,calcium, vitamins C, E, and D3, as well as dietary fiberand chondroprotectors.Its effectiveness was confirmed in the experimenton laboratory animals with modelled osteoporosis(ovariectomized female rats).The weight of the intact animals was mostly stablethroughout the experiment, with a slight increase fromthe 9th to the 15th day and from the 19th to the 26thday. The control animals, which received distilled water,gained weight during the entire experiment, especiallyfrom the 1st to the 12th day and from the 19th to the26th day. Two groups of experimental animals, whichreceived glucosamine + chondroitin and the drink basedon collagen fermentolysate, also gained weight from the1st to the 12th day and from the 15th to the 26th day.By the end of the experiment, the weight gainin the ovariectomized rats treated with distilledwater, glucosamine + chondroitin, and the collagenfermentolysate drink was 16.0, 12.5, and 14.3%,respectively. The weight gain in the intact group was5.3% (Fig. 4). Our data were consistent with the resultsof other studies [23]. Our findings were associated witha deficiency of estrogen that decreases the secretion ofleptin (a hormone with anorexigenic effect) from adiposetissue, thus leading to hyperphagia.Blood analysis showed a 29.6–60.5% increase inleukocytes in the ovariectomized animals, comparedto the intact group. However, statistical significancewas only registered in the group that received distilledwater. A statistically significant (P < 0.05) increase ofTable 4 Microbiological indicators of the functional productIndicator Contentin the powderStandard content,as in TR CU 021/2011Mesophilic aerobic and facultative anaerobic microorganisms, CFU/g, max. 8×104 1×105Mass of the product in which coliforms are not allowed, g not detected 0.1Mass of the product in which pathogenic bacteria, including salmonella, are not allowed, g not detected 25.0Mold, CFU/g, max. 30 200Figure 3 Contents of toxic elements in the functional productFigure 4 Weight changes in animals throughoutthe experimentMolecular weight, kDa0246810121416Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-Cys Val Met Phe Ile Leu Lys OPro Amino acids, g/100 g protein0.00.40.81.21.62.0lead arsenic cadmium mercuryToxic elements, mg/kgPermissible level (TR CU 021/2011)Content of toxic elements in the functional product43211 – rats on the standard diet (intact), 2 – rats on distilled water(control), 3 – rats on glucosamine + chondroitin (experiment 1),4 – rats on the collagen fermentolysate drink (experiment 2)360Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–36346.5% in lymphocytes was observed in the rats treatedwith glucosamine + chondroitin (reference sample),compared to the intact animals. All the ovariectomizedanimals had an increased content of granulocytes (upto 86.4%), compared to the intact group, although wefound significant variation between the experimentalrats within the group. Yet, the increased concentrationsof lymphocytes and granulocytes did not significantlyaffect their relative content.The control rats treated with distilled water hada statistically insignificant increase in monocytes of27.8% in relation to the intact group. This growth wasmore pronounced in both experimental groups (1 and 2),averaging 2.3 times (P < 0.05) in absolute terms and upto 47.8% (P < 0.05) in relative terms (Table 5).The cytometric analysis showed that bothsupplements to the diet (glucosamine + chondroitinand the functional drink) contributed to increasedcontents of leukocytes, granulocytes, lymphocytes, andmonocytes, although to a different extent. This meantthat they activated the blood immunity.According to the biochemical blood analysis(Table 6), the control rats had a significant increase,compared to the intact rats, in alkaline phosphatase,phosphorus, and calcium by 36.0, 2.8, and 15.6%,respectively. They also had a significant decreasein magnesium by 15.3%. We know that increasedconcentrations of these parameters in the blood areamong the diagnostic criteria for osteoporosis. However,the supplements of glucosamine + chondroitin andTable 5 Cytometric blood analysis at the end of the experimentParameter Standard diet(intact)Supplements to the dietDistilled water(control)glucosamine + chondroitin(experiment 1)drink based on collagenfermentolisate (experiment 2)Lymphocytes, 109/L 4.82 ± 1.47 5.48 ± 1.89 7.06 ± 1.47* 7.09 ± 2.73Leukocytes, 109/L 6.68 ± 2.14 8.66 ± 3.56 10.72 ± 2.95* 10.25 ± 4.14Mixture of monocytes, eosinophils,basophils and immature cells, 109/L0.46 ± 0.22 0.75 ± 0.35 1.04 ± 0.45* 1.01 ± 0.46*Monocytes, % 6.68 ± 1.20 8.54 ± 1.15 9.34 ± 1.89* 9.89 ± 2.22*Granulocytes, 109/L 1.40 ± 0.59 2.43 ± 2.15 2.61 ± 1.54 2.15 ± 1.22Lymphocytes, % 72.50 ± 5.23 64.93 ± 11.88 67.85 ± 11.03 69.93 ± 6.78Relative content of granulocytes, % 20.82 ± 4.73 26.53 ± 11.83 22.81 ± 9.68 20.17 ± 5.97* – significant difference from the intact group (Р < 0.05); ** – significant difference from the control group (Р < 0.05); + – significant differencebetween the experimental groups (Р < 0.05)Table 6 Biochemical analysis of blood serum at the end of the experimentParameter Standard diet(intact)Supplements to the dietDistilled water(control)Glucosamine +chondroitin(experiment 1)Drink based oncollagen fermentolisate(experiment 2)Proteins Total protein, g/L 76.50 ± 1.26 76.68 ± 2.62 80.52 ± 5.88 78.65 ± 4.70Albumin, g/L 42.18 ± 1.34 42.16 ± 1.91 42.11 ± 2.15 41.95 ± 1.03Low-molecularweightnitrogencontainingsubstancesCreatinine, μmol/L 65.09 ± 4.45 60.41 ± 3.55 60.13 ± 4.68 60.14 ± 2.99Urea, mmol/L 8.62 ± 1.60 9.65 ± 1.36 8.80 ± 1.16 8.89 ± 1.52Pigments Bilirubin (total), μmol/L 3.02 ± 0.42 2.63 ± 0.54 2.98 ± 0.45 2.77 ± 0.49Bilirubin (direct), μmol/L 2.12 ± 0.27 2.22 ± 0.45 2.18 ± 0.36 2.08 ± 0.31Enzymes ASAT, U/L 90.63 ± 9.70 95.80 ± 18.74 86.77 ± 9.79 94.86 ± 15.77ALAT, U/L 51.06 ± 16.58 48.11 ± 9.19 45.25 ± 6.54 44.48 ± 8.71Alkaline phosphatase, U/L 218.0 ± 41.0 339.2 ± 43.4* 194.9 ± 62.4** 208.4 ± 31.5**GGT, U/L 3.53 ± 1.30 3.55 ± 0.81 3.51 ± 0.88 3.15 ± 0.56LDH, U/L 189.8 ± 114.3 180.2 ± 87.8 186.7 ± 49.9 191.6 ± 85.0Lipids Cholesterol, mmol/L 2.18 ± 0.23 2.43 ± 0.58 2.73 ± 0.43* 2.60 ± 0.70Triglycerides, mmol/L 1.73 ± 1.23 1.45 ± 0.82 1.71 ± 1.21 1.22 ± 0.37Carbohydrates Glucose, mmol/L 11.33 ± 3.81 9.13 ± 2.32 8.66 ± 0.66 8.65 ± 1.61Inorganic compounds Calcium, mmol/L 2.82 ± 0.17 3.34 ± 0.19* 2.87 ± 0.17 2.85 ± 0.17Magnesium, mmol/L 1.18 ± 0.08 1.00 ± 0.05* 1.02 ± 0.08 1.11 ± 0.10**Phosphorus, mmol/L 2.85 ± 0.22 2.93 ± 0.24* 2.90 ± 0.29 2.86 ± 0.32* – significant difference from the intact group (Р < 0.05); ** – significant difference from the control group (Р < 0.05); + – significant differencebetween the experimental groups (Р < 0.05)361Aslanova M.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 354–363the functional drink decreased the activity of alkalinephosphatase to the intact level. This parameter in theexperimental groups was 35.5% (P < 0.05) and 31.0%(P < 0.05) lower than in the control group. It mightindicate a compensatory activation of the collagensyntheticfunction of osteoblastic cells in response toincreased activity of osteoclasts.The results of the biochemical analysis wereconfirmed by the determination of osteocalcin.Osteocalcin is the main non-collagenous protein ofthe bone matrix that is synthesized by osteoblasts. Itsconcentration in the blood reflects the metabolic activityof osteoblasts in bone tissue, since blood osteocalcinis synthesized, rather than released during boneresorption [24].There were no statistically significant changes inosteocalcin concentrations between the groups (Table 7).However, we found its increase of 15.0% (statisticallyinsignificant) in experimental group 2, which receivedthe functional product based on collagen fermentolysate,compared to the control.The contents of calcium, phosphorus, andmagnesium in the bone tissue of the animals under studyare shown in Table 8. As we can see, the control group ofovariectomized rats had decreased contents of calciumand magnesium (by 32.0 and 19.3%, respectively),compared to the intact group. They also had a significantincrease in phosphorus levels (by 22.0%).The experimental rats that received the functionaldrink had increased amounts of calcium and magnesium(by 25 and 3.0%, respectively), compared to the controlgroup. Although we also found a 7.0% decrease inphosphorus, it was not statistically significant. The ratiobetween calcium and phosphorus in experimental group2 was restored to 2.4:1.0 (2.5:1.0 in the intact group).According to our daily examinations, the generalcondition of all the animals was satisfactory interms of appearance, coat quality, and behavior. Тheexperimental animals looked identical to the controlgroup. Their coat was thick, tight, and glossy, with nosigns of fur loss. They were physically strong and hadno discharge from their natural orifices. Their limbs,motor functions, and behavioral reactions were normal.Their teeth were white, without plaque but with signsof abrasion. Their mucous membranes were pale, shiny,and smooth. The results of necropsy and macroscopicexamination did not reveal any hypofunction ordisplacement in internal organs (lungs, liver, spleen,stomach, kidneys, and pancreas). Their pulmonarypleurae, as well as pericardial and abdominal layers,were thin, shiny, and smooth. The hearts and aortas wereunchanged, and the vessels were moderately injected.Some animals in the control and experimental groupshad an enlarged uterus and mucus in the fallopian tubes.This might be associated with the involutional processesin their reproductive organs after surgery.We weighed the animals’ internal organs anddetermined their percentage in relation to the bodyweight. The results revealed no significant differencesfrom the physiological norms for the animals of thisspecies and age group.CONCLUSIONWe examined the quality characteristics of driedcollagen fermentolysate obtained from low-value byproductsof the meat industry (pork legs and ears, 1:1).Collagen fermentolysate contained 41% of peptidefractions with a molecular weight of 10 to 20 kDa. Italso had high contents of glutamic acid, aspartic acid,glycine, alanine, proline, leucine, and threonine. Theseamino acids stimulate cartilage and bone cells, restorejoint tissue, and are responsible for the production ofcollagen, elastin, and muscle tissue.Table 7 Osteocalcin concentrations in the blood serum of experimental ratsParameter Standarddiet (intact)Supplements to the dietDistilledwater (control)Glucosamine + chondroitin(experiment 1)Drink based on collagenfermentolisate (experiment 2)Osteocalcin, ng/mL 2.812 ± 0.569 2.618 ± 0.441 2.761 ± 0.522 3.049 ± 0.585Table 8 Bone mineral metabolism in ratsParameter Standard diet(intact)Supplements to the dietDistilled water(control)Glucosamine + chondroitin(experiment 1)Drink based on collagenfermentolisate (experiment 2)Calcium, mg% 15.3 ± 3.5 10.5 ± 2.6 14.0 ± 3.5 13.7 ± 3.4Phosphorus, mg% 5.70 ± 0.45 7.30 ± 0.58* 6.80 ± 0.54 5.60 ± 0.44Magnesium, mg% 7.80 ± 1.56 6.30 ± 1.26 6.80 ± 1.36 6.5 ± 1.3Ca:P ratio 2.5:1.0 1.5:1.0 2.0:1.0 2.4:1.0* – significant difference from the intact group (Р < 0.05); ** – significant difference from the control group (Р < 0.05); + – significant differencebetween the experimental groups (Р < 0.05)We developed a technology for a functionalproduct to prevent osteoporosis. Based on collagenfermentolysate, the formulation contained pumpkinpowder, dietary fiber, calcium, chondroprotectors, andvitamins E, C, and D3. The 80:20 ratio between proteinhydrolysate and pumpkin powder and the contents ofcalcium and vitamin D3 meeting 15 and 30% of the dailyintake, respectively, ensured a high nutritional value,functional effects, and good sensory characteristics ofthe product. The product is a powdered drink designedto mix with water. The microbiological and toxicologicalanalyses confirmed that the product complied with therequirements of TR CU 021/2011.The experiments on laboratory animals showed thatthe formulated product had an osteoprotective effecton the ovariectomized female rats. We found that thoserats which received the functional product had increasedcontents of calcium and magnesium in the bone tissue(by 25.0 and 3.0%, respectively) and a decreased contentof phosphorus (by 7.0%), compared to the control group.In addition, their calcium to phosphorus ratio wasrestored to 2.4:1.0 and the concentration of osteocalcin inthe blood serum increased by 15%.Our study makes a theoretical contribution tothe concept of safe bone homeostasis correction andproves that a functional drink based on connectivetissue protein can be used to prevent postmenopausalosteoporosis associated with hypoestrogenism.CONTRIBUTIONAll the authors were equally involved in, andtherefore are equally responsible for, developing thestudy concept, collecting and analyzing data, writingand editing the manuscript, and approving its finalversion.CONFLICTS OF INTERESTThe authors declare that there is no conflict ofinterest.