INTRODUCTIONIn recent years, unhealthy food habits and stressfullife style have significantly increased the risk of serioushealth disorders such as obesity, cancer, high bloodcholesterol, and coronary heart diseases. This has createdand increased demand for new health products withenhanced nutritional value. As a result, a number of researchhave been conducted in order to develop foodsthat are designed to improve digestive system health.One of these approaches is the development of functionalfoods using probiotics or prebiotics. Prebiotics can improvethe host health by stimulating the growth of beneficialbacteria in gastrointestinal tract [12]. Along withthe nutritional value of a functional product, its structuralproperties, such as water holding capacity (WHC) andsensory characteristics, and effective cost should be takeninto consideration [23].Inulin is a dietary fibre that has been approved byWHO as a safe prebiotic. It is a well-known and successfulfood ingredient in meat industry due to its uniqueability to enhance both taste and texture in various processedmeat products through binding water, forming geland mimics the oral tactile sensation of fat. The effectivenessof inulin has been approved in many investigationsin a wide range of processed meat products such asscalded sausages, canned meat products, meat balls, liverpâté, and fermented sausages [19].Konjac glucomannan, a neutral polysaccharidemade from the tuber Amorphophallus konjac, is anotherprebiotic that is known for its important technologicalproperties and its ability to improve health. USDArecently accepted the use of konjac as a binder in meatand poultry products. Studies suggested konjac hasthe ability to lower serum cholesterol, serum triglyceride,glucose, bile acid levels and laxative effect as well(Yang et al., 2017).Some investigations reported that appropriateamounts of konjac in the diet could help prevent diabetes178Safaei F. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 177–184and aid gradual weight loss. Several studies used konjacas a fat substitute, emulsifier, and gelling and thickeningagent in various meat products, such as low-fat frankfurter,bologna sausage, hot dogs, pepperoni, and summersausage [10]. Usually, the use of konjac in large amountsdecreases the firmness of meat products, and its combinationwith other ingredients such as inulin, starch or carrageenan,could moderate undesirable effect.Mixture design methodology is a new method to determinean effect of each ingredient in the formulationof processed meat products and demonstrate the result ofingredient interactions by applying reduced numbers ofexperimental trials [1].It should be noted that this is the first investigation oneffects of inulin and konjac on the physical and sensoryproperties of functional sausage. Hence, the objective ofthis research is to determine the influence of adding inulin,konjac, starch, and their mixtures on properties of sausagesusing the D-optimal mixture design and develop theoptimal formulation to produce a high quality sausage.STUDY OBJECTS AND METHODS1. Experimental design. To determine the optimumproportions of the prebiotic sausage formulation, weused Design-Expert (7.1.5) software. D-optimal designwas used with three components: konjac (K), inulin (I)and starch (S). The experimental design and the amountsof the relevant ingredients used are shown in Table 1.The component ranges were as follows: 0 < K < 0.5;0 < I < 5; and 0 < S < 5. Design-Expert software designed13 samples. Effects of inulin, starch, and konjacon properties of sausage were evaluated, and optimumcombination was determined. For optimization, dependingon the influence of each factor; the combination offactors that led to the best responses was determined.2. Sausage preparation. We prepared minced meatfor sausage according to a basic formulation. The mincedmeat consisted of 55% lean beef meat with fat contentof about 12.8 ± 1%, 10% soybean oil, 2.2% wheat flour,1.5% sodium chloride, 0.35% sodium polyphosphate,0.012% sodium nitrate, 0.02% ascorbic acid, 0.2% redpepper, 0.2% ginger, 0.1% savory, 0.2% garlic powder,and 17.418% water. All the ingredients were mixed in a3,000 RPM cutter (Talsa Bowel cutter 15, Spain). Then13 sausage samples were produced (5 kg each). Eachsample contained 4,750 g of the minced meat and variousproportions of konjac, inulin, and starch treatment(Table 1). The sum of starch, inulin, and konjac in eachsample was 5%. Sausages were stuffed into polyamidecasings and cooked in a steam oven at 80°C for 60 minuntil reaching an internal temperature of 72 ± 3°C. Inulin(Inulin Frutafit TEX®) and konjac flour was obtainedfrom Roosendaal (the Netherlands) and Shandong (China),respectively.3. Physical properties3.1. Water holding capacity (WHC). The WHC ofthe sausages was measured using the method describedby Asgharzadeh et al. and Méndez-Zamora et al. [5, 18].About 0.3 g of sausage was placed between two filtrepapers and then placed between two 12×12 cm plates.Four kg force was applied for 20 min. The released liquidsin the paper were considered as meat-free water.WHC was calculated using Eqs. (2) and (3). The experimentwas performed in triplicate for each sample.% of free water = [(Iw-Fw)/Iw]×100, (2)WHC = 100 – % of free water, (3)where Iw is the initial weight of the sample (0.3 g) andFw is the final weight.3.2. Cooking yield. A slice of raw sausage 3 mm inthickness was cooked on a hot plate at 160°C for 2 minaccording to the procedure described by Amini et al.[4]. Cooking yield was calculated using the initial andfinal weights and expressed in g/100 g the initial sampleweight. Three replicates were carried out for each sample.3.3. Frying loss. Frying loss was determined basedon the procedure described by Bengtsson et al. withsome modification [6]. Sliced cooked sausages, 1 cmin thickness, deep fat fried in a fryer (moulinex, DR5),maintained at 174°C, for 2 min until the center temperaturereached 72–73°C and then left to cool at room temperature.The frying loss was calculated by weighing thesamples before and after frying. The test was done intriplicate for each sample.4. Texture profile analysis (TPA). Texture profileanalysis (TPA) was evaluated using an InstronM350-10CT (500 N load cell, England, Rochdale). Thetextural parameters were determined according the Proceduredescribed by Bourne [7]. Textural measurementsincluded hardness and cohesiveness.5. Colour. Four samples from each formulation wereused to evaluate internal colour (cross-section) of thesausages. For that, we used 2 cm cross-sections of recentlycut sausage. The colour values of the samples weredetermined using a Chromo meter (CR-400, Minolta Co,Konica, Japan) with D65, 2° observer to objectively measureCIE Lab values (L* relative lightness, a* relativeredness and b* relative yellowness). Colorimeter calibratedwith white standard plate (L* = 94, a* = 0.3158,b* = 0.3322). The calculated results were expressed withmean value of these measurements.Table 1. Sausage samples with konjac (K), inulin (I), andstarch (S) in a three component constrained D-optimal mixturedesignSamples Ingredients, %K I S1 0.5 0 4.52 0.375 1.188 3.4383 0 0 54 0 2.5 2.55 0.5 0 4.56 0.5 4.5 07 0 0 58 0 5 09 0.125 3.688 1.18810 0.375 3.438 1.18811 0.5 2.25 2.2512 0.25 4.75 013 0.125 1.188 3.688179Safaei F. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 177–1846. Sensory evaluation. Sensory analyse was performedaccording to the international standards (ISO,1985) in the sensory laboratory at the National Nutritionand Food Technology Research Institute (NNFTRI). Privatestands under white fluorescent lights were preparedfor each panelist. Samples of each formulation were presentedrandomly for panelists. Tap water was available toclear the taste between samples. 15 panelists, 7 men and 8women, comprising of postgraduate students of food scienceand technology were asked to evaluate characteristicsusing a 9-point hedonic scale. The age of the panelistsranged from 20 to 40 years old. The panelists were trainedwith two training sessions in the product and terminology.Overall acceptability of the samples was scored as follows:1 (extremely dislike) to 9 (extremely like).7. Statistical and data analysis. Three equationmodels were fitted to each of the responses (Y) with theindependent variables:Linear model: Y= b1X1 + b2X2 + b3X3;Quadratic model: Y= b1X1 + b2X2 + b3X3 ++ b12X1X2 + b13X1X3 + b23X2X3; andCubic model: Y= b1X1 + b2X2 + b3X3 + b12X1X2 ++ b13X1X3 + b23X2X3+ b123X1X2X3,where X1is konjac, X2 is inulin, X3 is starch, and b is theregression coefficients calculated from the experimentaldata by multiple regression.All parametric tests were performed in triplicatefor each experiment and all the data demonstrated themean and SD (standard deviation). The physicochemicaland textural properties were studied using one-wayANOVA independently, and Duncan test was employedto determine differences between the experimentalgroups (p < 0.05). Sensory evaluation was analyzed bythe same software using Mann–Whitney U test. Correlationanalyses were conducted by using the Pearson correlationmodel where p < 0.05 was taken as significance.RESULTS AND DISCUSSIONFitting for the optimal model. The optimal modelwas fitted according to low standard deviation, low predictedsum of squares and high R-squared. P-values ofthe acceptable model were lower than 0.05.For frying loss, cooking yield, hardness andoverall acceptability, linear was found the bestmodel. For cohesiveness, a* and b* quadratic was adequatelyfitted. The model which best matched to waterholding capacity and L* were modified special cubic andspecial cubic, respectively.Water Holding Capacity (WHC). According to theregression coefficients in Table 3, all three componentsincreased WHC, however konjac had the greatest effect.Interestingly, the mixtures of inulin, starch, and konjacshowed a substantial effect on increasing the WHC ofsausages. This result is well correlated with results illustratedin Table 2, where samples no. 2 and 11 demonstratedthe highest WHC.The results revealed that, although adding inulinto the formulation of sausage could enhance WHC,the higher levels of inulin (more than 2.5%) decreasedthe WHC significantly. Sample no. 8 (contained5% inulin) demonstrated the least WHC. The synergeticeffect of konjac and inulin in absorbing wateris in agree with the study of Mendez-Zamora et al. Heinvolved inulin and pectin in the formulation of frankfurtersausages and showed that the addition of 15%inulin and pectin improved WHC [18]. Studies performedby [9] showed that konjac blend usually hadbeen used as multi-ingredient fat replacer in meat products.In addition, incorporation of konjac blend withcarrageenan and starch in low fat bologna increasedWHC, produced more stable gel matrix with higher cookingyield and more acceptable texture. López-Lópezet al. (described the type of fibers and quantity of theirpolysaccharides are the factors that influence waterholding capacity of product [17]. They mentionedlarge particles create open structures that enhance theproperties of hydration. Álvarez and Barbut also investigatedthe effect of beta-glucan (BG), inulin,and their mixture on the emulsion stability, and concludedcombination of BG and inulin compensa-Table 2. Cooking and sensory characteristics of experimental sausage samplesRUNWHCCookingyieldFrying loss Overall acceptabilityHardness CohesivenessL* a* b*1 63.78 ± 0.19 95.09 ± 0.16 21.05 ± 0.21 5.18 ± 0.11 22.63 ± 0.24 0.69 ± 0.01 39.84 ± 0.08 10.98 ± 0.09 15.56 ± 0.162 73.08 ± 0.21 93.58 ± 0.14 19.44 ± 0.17 5.57 ± 0.10 23.47 ± 0.21 0.59 ± 0.00 37.56 ± 0.08 8.39 ± 0.11 13.24 ± 0.193 59.37 ± 0.14 90.38 ± 0.14 17.59 ± 0.14 5.80 ± 0.10 21.10 ± 0.23 0.66 ± 0.01 38.01 ± 0.07 11.64 ± 0.08 16.19 ± 0.174 51.5 ± 0.22 89.76 ± 0.23 16.04 ± 0.12 6.27 ± 0.07 23.71 ± 0.24 0.62 ± 0.00 38.31 ± 0.08 12.27 ± 0.13 16.62 ± 0.165 63.77 ± 0.16 95.09 ± 0.16 21.03 ± 0.18 5.16 ± 0.09 22.63 ± 0.24 0.68 ± 0.00 39.83 ± 0.07 10.96 ± 0.07 15.59 ± 0.196 68.36 ± 0.28 93.86 ± 0.15 18.29 ± 0.21 6.06 ± 0.10 27.31 ± 0.22 0.62 ± 0.01 40.56 ± 0.07 10.58 ± 0.12 16.56 ± 0.197 59.37 ± 0.14 90.4 ± 0.16 17.62 ± 0.18 5.78 ± 0.07 21.09 ± 0.21 0.65 ± 0.00 38.01 ± 0.07 11.64 ± 0.09 16.19 ± 0.168 36.01 ± 0.22 89.07 ± 0.22 14.52 ± 0.15 6.83 ± 0.15 26.29 ± 0.21 0.71 ± 0.01 40.58 ± 0.07 18.25 ± 0.07 21.28 ± 0.189 54.16 ± 0.23 90.57 ± 0.17 16.20 ± 0.19 6.38 ± 0.12 25.33 ± 0.24 0.60 ± 0.00 38.88 ± 0.07 11.88 ± 0.14 15.97 ± 0.1910 72.10 ± 0.17 92.97 ± 0.15 18.07 ± 0.19 6.05 ± 0.15 25.84 ± 0.25 0.58 ± 0.02 38.11 ± 0.07 8.98 ± 0.12 14.18 ± 0.1811 82.96 ± 0.20 94.48 ± 0.15 19.62 ± 0.14 5.64 ± 0.14 25.00 ± 0.25 0.60 ± 0.02 36.78 ± 0.08 8.61 ± 0.14 14.34 ± 0.1512 52.84 ± 0.14 91.49 ± 0.22 16.42 ± 0.21 6.46 ± 0.15 26.82 ± 0.24 0.61 ± 0.00 40.66 ± 0.06 12.51 ± 0.13 16.64 ± 0.1413 62.25 ± 0.12 91.23 ± 0.14 17.71 ± 0.14 5.91 ± 0.15 22.74 ± 0.26 0.6 ± 0.010 37.82 ± 0.07 9.44 ± 0.12 13.93 ± 0.19180Safaei F. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 177–184ted undesirable effect of fat reduction by increasingWHC [3]. Liu et al. also prepared konjac-egg white proteingels and determined that konjac could significantlyimprove the water retention capacity [15].Cooking yield and frying loss. The results in Table 3revealed that konjac with its positive coefficient had significantly(p < 0.05) increased cooking yield, while inulinand starch with their negative coefficient decreased thisparameter in the product. The samples no. 1 and 5, whichcontained highest amount of konjac (0.5% konjac, 4.5%starch, and 0% inulin), showed the highest cooking yieldand frying loss. As one can see in Table 1, two pair sam-Table 3. Regression coefficients and correlation for the adjusted model to experimental data in D-optimal mixtures design forphysical properties, textural parameters, color parameters, and sensory analysisParameter K I S KI KS IS KIS Pred-R2WHC 103.49 36.00 59.37 284.30 – 15.27 682.63 0.9997Cooking yield 137.18 89.06 90.40 – – – – 0.9951Frying loss 10.40 2.90 3.51 – – – – 0.9939Overall acceptability –0.01 6.99 5.99 – – – – 0.9579Hardness 36.44 26.31 21.10 – – – – 0.9908Cohesiveness 16.15 0.70 0.65 –18.13 –16.89 –0.23 – 0.8950L* 6.44 40.58 38.01 37.70 55.36 –3.95 –129.30 0.9961a* 631.78 18.26 11.64 –767.03 –696.53 –10.72 – 0.9981b* 783.11 21.27 16.20 –899.03 –859.00 –8.41 – 0.9923Fig. 1. Contour plots for effectы of konjac (A), inulin (B)б and starch (C) on water holding capacity (WHC), cooking yield, fryingloss, hardness, cohesivenessб and overall acceptability of prebiotic sausage.181Safaei F. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 177–184ples are similar: one pair is samples no. 1 and 5, anotherone is the samples no. 3 and 7. These compositions weredefined by Design Expert software to check the repeatabilityof findings. As it was expected similar compositionshave displayed comparable results for cooking andsensory characteristics in Table 2, indicating the repeatabilityof findings.Several studies suggested to use konjac in combinationwith other hydrocolloids. Emir et al. discussed thatweak junction zones in konjac made it susceptible to heat,and its interaction with other hydrocolloids caused tightsjunction which made it resistant to cooking or frying [11].According to regression coefficients (Table 3), inulincaused decrease in cooking and frying loss. The sampleno. 8 (with the maximum level of inulin) displayed theleast cooking yield and frying loss. This data is similarto that of Afshari et al. who indicated although inulin isable to increase WHC and decrease frying loss, but, athigher amounts, it reduced the moisture retention andcooking yield probably due to its porous structures andinability to form a tight gel [1].Texture profile analysis (TPA). As the hardnessanalysis showed, konjac had a strong effect on the hardnessof sausage, inulin also increased it, while starch, onthe contrary, reduced the hardness of the product.Several studies indicated that the addition ofkonjac into the food matrix increased the hardnessof products but it depends to many factorsthat should be taken into consideration. Thesefactors are the molecular weight and particularly thetype of konjac (flour or as hydrolyzed), pH of a foodsystem, presence of salts, and an amount of incorpora-Fig. 2. Contour plots for effectы of konjac (A), inulin (B), and starch (C) on lightness (L*), redness (a*), and yellowness (b*)of prebiotic sausage.ted konjac and other food ingredients, specially gellingagents. All these studies are in agree that increasein amount of konjac increase the hardness that may notbe accepted by consumers. Hu et al. reported that konjacglucomannan (KGM) affected functional propertiesof egg white protein and increased hardness,chewiness, and springiness of the gel samples at a certainconcentration [13]. The investigation conductedby Emir et al. indicated that the bigger molecularweight of KGM caused the highest hardness and closelythe lowest springiness, which had negative effect onthe choice of panelists [11]. Akesowan reported thatincreasing of NaCl resulted increase in links betweenkonjac/κ-carrageenan and konjac/gellan, leading tothe increment in the hardness of the produced gel [2].Purwandari et al. used konjac a noodle formulation.They found that the hardness and adhesivenessof noodle significantly increased (p < 0.05) and becamethree times harder than standard Chinese or Japanesewheat noodle [21]. The researchers also indicatedthat an increase in proportion of water in pregelatinisedflour led to increased harness in konjac noodle.Several studies also determined that the use of powderedinulin resulted in higher moisture loss during cooking.This can affect the texture of a product and increasedhardness of burgers, frankfurter sausages anddry-fermented chicken sausages. [1, 18 and 19]. These resultsare in a good agreement with the results of this study.Another texture parameter related to meat products iscohesiveness. Adhesiveness and cohesiveness are parametersthat play an important role in handling of sau182Safaei F. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 177–184sages, particularly for the slicing of these products. Ifproducts are too adhesive or cohesive, they become undesirablysticky, and it cannot be easily to cut [20]. In thecurrent investigation konjac showed a significant positiveeffect on cohesiveness of probiotic sausage. On thecontrary, combination of konjac with inulin or starch reducedthe undesirable effects of using konjac alone andmaintained the appropriate adhesiveness and cohesivenessof sausages with improved textural properties. Thestudy [9] documented that when konjac was used as amulti ingredient in the formulation of meat products, unwantedhardness and cohesiveness decreased significantly.The researchers suggested to incorporate konjac incombination with other hydrocolloids Purwandari et al.also confirmed that konjac had a substantial effect on theincrease of adhesiveness and cohesiveness of noodle:konjac noodle was about ten times more cohesive andsticky than wheat flour noodle [21].Colour. As shown in Table 3 and Fig. 2, all threecomponent separately increased the lightness of theproduct, while their combination in special cubic modelcaused decrease in L* value in the sausages. The resultsindicated that inulin was meaningfully (p < 0.05) more effectivein enhancing the lightness compared to starch andkonjac. The samples no. 6 and 8, which containing thehighest amount of inulin, illustrated the most lightness.According to Table 3, konjac demonstrated a significant(p < 0.05) positive effect on a* and b* values causingmore reddishbrown product, while its combinationwith starch or inulin decreased a* and b*. Trespalaciosand Pla reported that if when myoglobin and fat contentwas maintained constant, the color of formulated productswas mostly influenced by many factors, includingadditive ingredients [24]. In the present study, as theprotein and fat content was invariable, the color was influencedmainly by mixing ingredients. Amini et al. mentionedkonjac led konjac to a more reddishebrown of aproduct by its susceptibility to Maillard browning [4].Jiménez-Colmenero et al. also indicated that the additionof konjac in frankfurter sausage caused decrease(p < 0.05) in lightness (L*) and an increase (p < 0.05) ofyellowness (b*), compared to other samples [14]. The resultsof another investigation, conducted by Ruiz-Capillaset al., are in agreement with the present study thatkonjac gel affected color parameter of sausages throughdecrease in L* and increase in yellowness (a*) [22]. Delgado-Pando et al. observed less red, paler (p < 0.05), andyellower pâtés as a result of adding konjac [10].Sensory analysis. The experimental results obtainedfrom the regression coefficient values of overall acceptability(Table 3 and Fig. 1) displayed that increasein the proportion of konjac had a significant (p < 0.05)negative effect on the overall acceptability of the product.Inulin showed a positive effect on the acceptabilityof the product, while starch was not significantly effective.Formulation 12 (contained 0.25; 4.75; and 0%konjac, inulin and starch, respectively) and formulation9 (0.125; 3.68; and 1.18%) demonstrated the highestoverall acceptability score.Results obtained by sensory analysis highlightedthat adding konjac in the amount of up to 0.2% couldimprove the appearance of sausage. On the contrary,increase in the amount of konjac (more than 0.2%) decreasedthe overall acceptability significantly (p < 0.05).The results emphasized that hardness and cohesivenessare the factors that significantly influence overall acceptability.Increase in proportion of konjac (more than0.2%) can make the sausage harder and more cohesivethan standard sausage which may not be acceptable byconsumers. In the other words, consumers would not accepta product with extreme hardness or cohesiveness.Another explanation is that high amount of konjac probablyenhances its typical fishy taste/odours. These findingsare in a good agreement with the results of Purwandariet al. who reported that the addition of konjac glucomannancould improve sensory perception of wheat noodle,while a high level of this ingredient reduced preference,since noodle became too sticky [21]. Lin et al. also observedthat 1% konjac in reduced-fat frankfurter sausagesled to higher scores of sensory overall acceptability [15].Fig. 3. Desirable plot for optimum formulation.183Safaei F. et al. Foods and Raw Materials, 2019, vol. 7, no. 1, pp. 177–184Liu et al. also assumed that functional propertiesof food products could be controlled by adding smallamounts of KGM without causing undesirable sensorychanges [16].On the contrary, inulin showed positive coefficient onoverall acceptability of sausages and an increase in portionof inulin improved the product flavour. Menegas etal. represented incorporation of inulin (maximum levelof 7.5%) in reduced-fat sausages made the product morefavorable and acceptable by consumers [19].Mixture proportion optimization and desirabilityfunctionThe optimisation was done in order to access the optimalamount of each component that had an excessiveeffect on quality properties of the sausages. The predictedvalues of the responses are shown in Fig. 3. Ouraims were to maximize overall acceptability and cookingyield of the sausages, minimise frying loss, and, at thesame time, to maintain WHC, hardness, and cohesivenesswithin normal range. Having all these criteria taken intoconsideration, we found that optimal amounts of inulin,starch, and konjak were 2.09; 2.76; and 0.146%, respectively(Fig. 3). The selected mixture achieved 0.858 desirabilityscore. As the desirability value between 0.8 and1.0 is recognized as acceptable and excellent product, theformulation with 0.858 desirability value was selected asoptimal formulation that could provide valuable nutritionaland technological properties.CONCLUSIONIn conclusion, the development of functional foodsopens up new possibilities for the food industry andconsumers. The development of healthier sausage withprebiotics inulin and konjac is a promising direction ofresearch. The physicochemical and sensory characteristicsof the prebiotic sausages are conditioned by the formulation.The study demonstrated that the sausage contained0.146; 2.09; and 2.76% konjac, inulin, and starch, respectively,has high quality and sensorial properties.CONFLICT OF INTERESTThe authors declare that there is no conflict of interests.ACKNOWLEDGEMENTSWe are thankful to Tehran Meat Products Companyfor their help in sausage manufacturing.FUNDINGThis research was financially supported by NationalNutrition and Food Technology Research Institute(NNFTRI) of Iran.