Determining the effects of encapsulated polyphosphates on quality parameters and oxidative stability of cooked ground beef during storage

The shelf life of meat and meat products is significantly reduced by lipid oxidation which also leads the generation of a large number of by-products that can have negative effects on consumer health. On this regard, controlling lipid oxidation is a great deal of importance in the production of healthy meat and meat products to meet demands of consumers who claim quality and healthy food (Min & Ahn, 2005).


Introduction
The shelf life of meat and meat products is significantly reduced by lipid oxidation which also leads the generation of a large number of by-products that can have negative effects on consumer health.On this regard, controlling lipid oxidation is a great deal of importance in the production of healthy meat and meat products to meet demands of consumers who claim quality and healthy food (Min & Ahn, 2005).
Lipid oxidation starts in the membrane phospholipid fractions of muscle cell membranes and accelerates during process and storage of meat and meat products (De La Dssa, 2009).As a result of lipid oxidation, the nutritional value of the meat and meat products is reduced as far as development of undesirable taste, color, smell and toxic compounds (Ulu, 2004;Ceylan et al., 2007).
Many strategies are implemented in meat industry to prevent lipid oxidation, inhibit undesirable changes, increase shelf life and protect human health.The use of antioxidants is the most common strategy in meat and meat products technology (Nassu et al., 2003).
Polyphosphates have strong antioxidant effects that can retard the oxidation reactions by chelating metal ions (Sickler, 2000;Bektas, 2009;Fonseca et al., 2011).At the same time, the polyphosphates have many other beneficial functional properties such as pH buffering during the production of cooked meat products, reduction of cooking loss, increasing water-holding capacity, improvement of the textural and sensory characteristics of muscle foods (Sickler, 2000).Addition of alkaline polyphosphates during the preparation of meat products provides an increased water-holding capacity and product yield by raising the pH and enhancing the swelling properties of muscle proteins.However; acid polyphosphates lowers the pH and negatively affect the water-holding capacity by shrinking the meat proteins (Yapar et al., 2006;Anar, 2012;Shao et al., 2016).Polyphosphates also increase the consumer acceptability of the product by promoting the color formation and stability via pH buffering effect (Baublits et al., 2005).Polyphosphates have a synergistic effect with salt to enhance the textural properties by improving extraction of myosin (Hsu & Yu, 1999;Cheng & Dckerman, 2003;Gadekar et al., 2014;D'Flynn et al., 2014).Even though polyphosphates are not known as antimicrobial ingredients, they have antimicrobial properties at a certain level by sequestering metal ions such as Ca, Mg, and Fe that play role in microbial growth (Akhtar et al., 2008;Long et al., 2011;Aksu & Alp, 2012).
The chelating ability of polyphosphates increases with increase in pH, temperature and the chain length of polyphosphate (Marsh, 1992).However, the antioxidant potential of polyphosphates is partially reduced by endogenous phosphatase enzymes in the meat due to hydrolysis of polyphosphates into orthophosphates (Yamazaki et al., 2010).Hydrolysis of polyphosphates into orthophosphates causes problems in maintaining the oxidative stability of cooked muscle foods during storage since a low antioxidant potential of orthophosphates.Even though the application of heat totally inactivates the enzymes, significant amounts of polyphosphates are hydrolyzed prior to heat processing and they are not utilized effectively as an antioxidant.Therefore, it is critical to protecting the polyphosphates from hydrolysis until phosphatase enzymes are totally inactivated by heat processing (Kılıç et al., 2014).
Encapsulation technology is defined as the coating of solid, liquid or gaseous food ingredients, enzymes, cells, microorganisms and other substances by a coating materials such as oil, protein or carbohydrate (Gouin, 2004;Madene et al., 2006).The encapsulation technology has been used successfully in the food industry to maintain stability a functional property of encapsulated substance by protecting them against moisture, heat and other harsh conditions (Kılıç et al., 2014).
On this study, the use of encapsulated polyphosphates and their combinations on oxidative stability and physicochemical properties of cooked ground beef during storage was investigated.
Vacuum packaged beef muscle was stored frozen at -18 °C until used.After thawing, the beef was ground (9.5 mm), mixed and then again ground (3.2 mm).1% sodium chloride and 10% water was added to ground meat (meat weight basis).Then the ground beef was separated equal amounts for each experimental group.Test ingredients (polyphosphates) were added according to the experiment formulation of each group.10 different treatment groups including encapsulated and un-encapsulated STP or SPP or their combinations were prepared (Table 1).The whole experiment was replicated (two times) on separate production day and the analysis was carried out in duplicate for each experimental replication.The experimental groups were subjected to the cooking process in centrifuge tubes containing 45 g ground meat.After the tubes were placed in water-bath at 60 °C, the temperature of the water-bath was set at 85 °C.The standard number tube was placed in the water-bath during each experiment and the final internal cooking temperature was monitored with the thermocouple placed in the geometric center of extra tube with 45 g ground meat.Cooked samples were stored at 4 °C for 7 days and they were analyzed for dependent variables.

Cooking loss analysis
The weight of raw ground beef used for each test group was registered.The weight of cooked ground beef was also determined after the samples were cooled to approximately 25 °C.Cooking loss was calculated according to the following Equation 1.

( )
weight of raw ground beef -Cooking loss: / weight of raw ground beef * 100 weight of cooked ground beef

pH analysis
5 g sample was taken from each group and homogenized 1 min in 50 ml of distilled water using homogenizer.pH measurements were performed using glass electrode (FC 200,Hanna Onstruments,Germany) which is calibrated with the buffer solutions pH 4.0-7.0.

Color analysis
The color measurements were performed with Minolta Colorimeter (Model CR-200, Olluminant D65, Minolta corp., Ramsey, NJ, U.S.A.) and COE L* a* b* values were determined (Hullberg & Lundström, 2004).Colorimeter was calibrated using its own standard before the measurements.

Soluble orthophosphates analysis
The amount of soluble orthophosphate was determined with modified procedure of Molins et al. (1985).Cooked ground beef (2 g) samples were taken, mixed with 18 ml of deionized water and homogenized 13500 rpm for 10 s.Homogenized samples were kept at 4 °C for 30 min.Samples were filtered through Whatman No. 1 filter paper and then 1 ml filtrate was centrifuged with 1 ml 10% TCA at 4000 rpm for 10 min.After centrifugation, 0.1 ml supernatant was diluted with 0.5 ml distilled water and on the mixture was mixed with 1 ml of a color solution (6N sulfuric acid+distilled water+2,5% ammonium molybdate+10% ascorbic acid, volume 1:2:1:1, respectively).Absorbance was read at 690 nm within 1 h after the mixture was incubated at 37 °C for 10 min.The amount of soluble orthophosphate was determined by a standard curve prepared using potassium phosphate.

TBARS analysis
TBARS analysis was carried out according to Kılıç & Richards (2003).On this method, the trichloroacetic acid (TCA) extraction solution is added into EDTA and the propyl gallate to prevent the formation of TBARS during analysis.1 g of beef sample was mixed with 6 ml extraction solution, homogenized for 15 s and filtered through Whatman No.1 filter paper.The filtrate (1 ml) was vortexed with 1 ml of thiobarbituric acid (TBA, 98%).Then the mixture was heated at 100 °C for 40 min and centrifuged at 2000 rpm for 5 min after cooling.The absorbance values were determined at 532 nm against a blank including 1 ml TCA extraction solution and 1 ml TBA solution.On order to achieve accurate measurement, each filtrate was measured two times, and their average value was taken as the final reference value.TBARS values were calculated by multiplying absorbance values with coefficient obtained from a standard curve.A calibration curve was constructed for each run using tetramethoxypropane as the standard.

Lipid hydroperoxide analysis
The method which was explained by Shantha & Decker (1994) was used for the lipid hydroperoxide analysis.On this method, 1g of the sample was homogenized within 5 ml chloroform/methanol (1:1) in 30 s and vortexed for 30 s again after adding 3 ml of 0.5% NaCl.The mixture separates into two phases after centrifuging for 10 min. 2 ml were taken from the lower phase and mixed with cold chloroform/methanol (1.3 ml; 1: 1). 25 μl of ammonium thiocyanate (4.38 cm) and 25 μl of iron (OO) chloride (18 mM) was added to assay the lipid hydroperoxide (Shantha & Decker, 1994).Samples were incubated at room temperature for 20 min before determining the absorbance values at 500 nm and the standard curve was prepared using a cumene hydroperoxide.

Statistical analysis
The eleven different experimental groups were produced and the whole study was replicated two times and the analysis was carried out with two replications.The differences identified in the analysis results were calculated by using Statistical Package for the Social Sciences (SPSS) 18.The data obtained cooking loss measurement results were evaluated by one-way analysis of a variance technique.The data obtained TBARS, lipid hydroperoxide, soluble orthophosphate, pH and color measurement results were applied repeated measures analysis of a variance technique.Tukey test was used to determine the difference between the mean of the groups.

Cooking loss
The results presented (Table 2) the lowest CL values were obtained in samples manufactured with STP and the highest CL values were determined in samples incorporated with SPP and control group (p<0.05).Lee et al. (1998) reported that STP significantly increased the cooking yield compared to SPP in restructured polyphosphates beef.Similarly, Cheng & Dckerman (2003) stated that added phosphate in conjunction with the tumbling process created a more uniform product and the higher cooking yield by increasing the ionic strength.Ot was demonstrated that the addition of phosphate decreased the CL by increasing water-holding capacity and pH (Villamonte et al., 2013).
The results showed that there was no significant difference in terms of CL values among the samples containing uSTP or eSTP or their combination.However, this was not a case in the samples produced with SPP.CL values of samples containing 0.5% eSPP was significantly lower than the samples containing a combination of uSPP and eSPP (p<0.05).Results indicated that the use of uSTP or eSTP in combinations with uSPP or eSPP resulted in higher CL values in samples compared to those produced with either uSTP or eSTP (p<0.05) and did not contribute to reducing CL values obtained in samples incorporated with uSPP or eSPP except for eSTP+eSPP group.As far as the combinations of STP and SPP are concerned, a combination of eSTP and eSPP provided lower (p<0.05)CL than the other combinations groups except eSTP+uSPP which had similar CL values.Sickler et al. (2013a, b) reported that the addition of encapsulated or un-encapsulated STP reduced CL values in beef and turkey meat.Furthermore, Kılıç et al. (2014) also reported that addition of encapsulated or un-encapsulated STP had the lowest CL values in ground chicken and ground beef.Kılıç et al. (2015) also stated that the use of uSPP or eSPP in ground chicken and ground beef increased the CL values.The authors stated that increased CL as a result of the use of SPP was associated with the lowering of the meat pH.
Ot is well demonstrated that water-holding capacity of the meat depends on pH.Water-holding capacity is lowest when meat pH get close to isoelectric point of the meat proteins (Genccelep, 2008).Ot has also been stated that alkaline polyphosphates increase water-holding capacity and decrease CL by raising the pH of meat.(Sickler et al., 2013a).Ot has also been reported that water-holding capacity is enhanced by STP due to improving the solubility of the meat protein (Hsu & Yu, 1999).

pH
Dn the average pH values obtained during a storage period of samples were illustrated in Table 2. Ot was found that polyphosphate type is effective on the change in pH (p<0.05).While STP increased pH, SPP caused a decrease in pH compared to control (p<0.05).Ot was previously stated that the alkaline polyphosphates tend to increase the pH and improve water-holding capacity (Cheng & Dckerman, 2003;Cheng et al., 2007;Roldán et al., 2014;Chmiel et al., 2015).
Results indicated that the storage day did not appear to influence pH values for all treatment groups.pH values were almost constant during a storage period for each treatment group.Likewise it was not found any significant difference among the samples containing uSPP or eSPP or their combination.As far as the combinations of STP and SPP are concerned, pH did not show significant differences among these combination groups.This means that the encapsulation had no effect on final pH of a combination groups.Kılıç et al. (2014) reported that the encapsulated polyphosphates did not create a significant impact on the final pH due to completely release from hydrogenated vegetable oil during the cooking process.Dur results are in agreement with previous studies about the encapsulated polyphosphates effects on meat pH (Kılıç et al., 2015;Sickler et al., 2013a;Kılıç et al., 2016a, b).pH values of STP and SPP combinations were found to be a lower than groups formulated with solely uSTP or eSTP and generally similar with control and only uSPP or eSPP incorporated groups.

Color
The results of COE L*, a*, b* values were presented in Table 3. Results revealed that the use of uSTP resulted in lower (p<0.05)L* values while the samples with uSPP or eSPP or eSTP had similar L* values compared to control during each storage time.On addition, the use of uSTP or eSTP or their combination did not create any difference in L* values among these three treatment groups during storage.This was also a case as far as SPP was concerned.This results demonstrated that the encapsulation of polyphosphates had no significant effect on L* values of ground meat.Furthermore, no significant L* value differences were observed among the groups formulated with a various combination of uSTP or eSTP with uSPP or eSPP.Results showed that L* values of all treatment groups were quite constant during the whole storage period.Lee et al. (1998) reported that the use of STP and SPP decreased L* and b* values but increased a* values of raw beef rolls.However, in cooked beef rolls, the authors stated that there were no significant differences among treatment groups in L*,a*,b* values.
Results indicated that cooked ground beef formulated with uSPP or eSPP had lower (p<0.05).a* values on day 0 and 1 compared to control which had similar a* values with the samples incorporated with uSTP or eSTP.However, on 7-d display, there were no significant differences between control and the samples with uSPP or eSPP or their combination while higher (p<0.05)a* values were determined in samples containing uSTP or eSTP or their combination.This result may be explained by a superior buffering effect on myoglobin.On addition, there was no significant difference regarding a* values among the groups containing uSPP or eSPP or their combination.The same result was also obtained in the samples produced with uSTP or eSTP or their combination.Regarding combinations of STP and SPP, the highest a* values were observed in the group formulated with uSTP and uSPP (p<0.05).Rest of the combinations (uSTP+eSPP, eSTP+uSPP, eSTP+eSPP) had similar a* values which were not found to be different from those determined in the control group.Furthermore, a decreasing trends in the a* values during storage were observed in control, eSPP, uSTP+eSPP, eSTP+uSPP and eSTP+eSPP groups (p<0.05).Nevertheless, the a* values in rest of the treatment groups were quite stable during a 7-d of storage.
Results indicated that there were no significant b* values differences among treatment groups on day 0 except uSTP+eSTP group which had lower b* values than those of uSPP, eSPP and uSPP+eSPP groups.However, the treatment effects on b* values were more evident at the end of the storage (p<0.05).On general, the control and groups formulated with uSPP or eSPP or their combinations tend to have increasing trends in b* values during storage and had higher b* values at the end of the storage compared with groups incorporated with uSTP or eSTP or their combination.Baublits et al. (2005) reported that the use of STP resolved the deterioration of meat color caused by 2% added NaCl.Kılıç et al. (2014) reported that the use of eSTP reduced COE L* and b* values and increased a* values.Likewise, the authors stated that the use of eSPP caused an increase in L* and b* values and a decrease in a* values.
The study results indicated that the use of uSTP or eSTP or their combination caused a decrease in L* and b* values and increase in a* values of cooked ground beef at the end of the storage period.Similar results were reported previously (Baublits et al., 2005;Kılıç et al., 2014).

Soluble orthophosphate content
Soluble orthophosphate results were shown in Table 4.The soluble orthophosphate contents of all treatment groups did not show any significant change during 7-d of storage period.The lowest soluble orthophosphate values were determined in the control group.Addition of polyphosphates into the meat was found to increase the amount of soluble orthophosphate in the samples (p <0.05).This observation was also supported by Lee et al. (1998).Authors reported that the amount of soluble orthophosphate was increased as increasing quantity of phosphate added to meat.A significant difference was not found among the samples containing uSTP or eSTP or their combination on each day of the storage period.However, the use of eSPP significantly reduced orthophosphate content compared to that of uSPP (p<0.05) as far as all the combinations were concerned, orthophosphate content did not show significant variations among combination groups.This result showed that SPP can be effectively protected from phosphatase enzyme activities due to the encapsulation process, but this was not evident for STP.Kılıç et al. (2014) encapsulation process was effective to reduce soluble orthophosphate content in the ground meat.On addition, Kılıç et al. (2016a) predicted that the use of STP in meat contributed to more orthophosphate formation compared to that of SPP due to increased pH by STP.

TBARS
The average of TBARS values was presented in Table 5. Ot was determined that added STP and SPP was restricted TBARS formation effectively compared to the control group (p<0.05).Lee et al. (1998) reported that transition metals such as iron and copper and heme compounds play an important role in the oxidation of polyunsaturated fatty acids in the meat and meat products.Researchers stated that polyphosphate inhibits lipid oxidation by forming chelates with metal ions.The authors indicated that the polyphosphate reduced TBARS values in the cooked meat products by especially binding of non-heme iron.Dther previous studies also indicated that the addition of polyphosphates into meat was an effective strategy to enhance the oxidative stability of meat products by reducing the TBARS formations (Cheng & Dckerman, 2003;Hsu & Sun, 2006).
TBARS results revealed that the control group had higher (p<0.05)TBARS values on processing day than the rest of all treatment groups which were not different from each other.On general, there was a gradual increase in TBARS values of treatment groups during storage period (p<0.05).At the end of the storage period, the lowest (p<0.05)TBARS values were determined in the samples formulated with only eSPP or combinations including eSPP (uSPP + eSPP, uSTP + eSPP, eSTP + eSPP).This result showed that lipid oxidation inhibition was effectively enhanced by the use of eSPP.This result showed that increasing the amount of eSPP added to a meat system provide an increase in the amount of active phosphate (the amount of pure phosphate added to the meat), leading to have more effective lipid oxidation inhibition.Dn the other hand, uSTP or eSTP or their combination was found to be less effective in controlling TBARS formation compared to SPP counterparts.However, these groups still had lower (p<0.05)TBARS compared to control.On addition, no significant TBARS differences existed among these groups.This means that the encapsulation of STP did not provide any significant benefit to having a further reduction  in TBARS formation.According to the obtained results from this study, it can be concluded that eSPP is more effectively limited lipid oxidation in cooked ground beef.Kılıç et al. (2014Kılıç et al. ( , 2015) ) reported that TBARS values of ground meat were reduced with the use of encapsulated polyphosphates.Kılıç et al. (2016b) also reported that the highest oxidative stability was accomplished in the ground meat containing eSTP and eSPP.However, researchers did not test a combination of different polyphosphate types in their study.

Lipid hydroperoxides
The results for lipid hydroperoxide were presented in Table 6.Similar to TBARS results added STP and SPP caused to reduction in lipid hydroperoxide values compared to control group (p<0.05).Although the lower (p<0.05)LPD values were obtained in the samples containing eSTP compared to those produced with eSPP, there were no significant LPD differences among all treatment groups on processing day.As expected, a gradual increase (p<0.05) in LPD values were observed during a 7-d storage period in all treatment groups except eSPP, uSPP+eSPP, uSTP+eSPP and eSTP+eSPP groups where LPD values were quite stable during the whole storage period.Results indicated that the lowest LPD values were obtained in the same treatment groups (eSPP, uSPP+eSPP, uSTP+eSPP and eSTP+eSPP) at the end of the storage period (p<0.05).The findings showed that the rest of treatments provided also a significant reduction in LPD values compared to control (p<0.05).Kılıç et al. (2015) reported that the values of lipid hydroperoxide in the ground meat with encapsulated polyphosphates were significantly lower than those without any polyphosphate addition.

Conclusion
The findings of the present study demonstrated that SPP was the most effective type of polyphosphate in the limiting lipid oxidation development in the cooked ground beef during storage and the encapsulation process strongly enhanced the effectiveness of SPP on lipid oxidation inhibition.On addition, results indicated that the same level of lipid oxidation inhibition can be accomplished with the use of eSPP or uSPP + eSPP or uSTP + eSPP or eSTP + eSPP.Based on the study results, the use of eSTP + eSPP combination in ready to eat meat product formulation is suggested to the meat industry to have extended shelf life.However, it is important to keep in mind that the eSTP+eSPP combination may partially reduce a beneficial effect of STP on cooking loss.

Table 1 .
Coding for the ten polyphosphate treatments and control group evaluated.
C: control group, STP: sodium tripolyphosphate, SPP: sodium pyrophosphate, e: encapsulated.u: un-encapsulated; a The amount of polyphosphate added on a meat weight basis.

Table 2 .
Cooking loss and pH values of treatment groups.

Table 3 .
COE L*, a*, b* color values of treatment groups during storage period.

Table 4 .
Soluble orthophosphate content (μg/g) of treatment groups during storage period.

Table 5 .
TBARS results (µmol/kg) of treatment groups during storage period.