Quality characteristics of low-fat chicken sausages formulated with whey protein isolate and guar gum at different storage conditions

ABSTRACT We aimed to explore the physiochemical and microbiological attributes of cooked chicken sausages, incorporating sesame seeds, whey protein isolate (WPI), and guar gum (GG) as non-meat replacers. Both control and sausage samples were subjected to storage under refrigeration (4°C ±1°C) and frozen (−8°C ±1°C) conditions. The pH values of the chicken sausages exhibited a significant upward trend (p < .05) under both refrigeration and frozen storage conditions. The formulated sausages exhibited elevated average cooking losses of 13.26% (p < .05). Importantly, peroxide values remained below the acceptable threshold for oily food. Furthermore, the inclusion of WPI and GG in the sausages led to a lower average water activity value of 0.55 Aw (p < .05) under frozen conditions than the water activity of 0.90 Aw (p < .05) under refrigerated conditions. The total viable count was not significantly affected (p > .05) by the addition of WPI and GG under freezer conditions. Consequently, it can be inferred that the cooked chicken sausages can be safely stored under frozen conditions for a duration of 60 days.


Introduction
According to the Brunei Public Health (Food) Act 2001, [1] chicken sausage is classified as a processed meat product with a fat content not exceeding 40%.Fat plays a crucial role in stabilizing the meat emulsion, and its absence may result in an undesirable mouthfeel, affecting the texture, juiciness, and other sensory attributes of the sausage.However, many sausage products on the market contain high levels of sodium and saturated fat.For example, a low-sodium pork sausage contains 35% saturated fat and 23% sodium, a reduced-sodium turkey sausage contains 19% saturated fat and 25% sodium, and a beef sausage contains 80% saturated fats and 52% sodium.A prior study on the physicochemical properties of commercially available chicken sausages in Malaysia reported that the fat and sodium contents were within the ranges of 4.91%-18.48%and 6.52-10.36mg/g, respectively. [2]Excessive consumption of sodium and saturated fat poses health risks, including the potential for heart disease, stroke, and hypertension. [3]To address these concerns, the food industry has explored various approaches, including the use of non-meat fats from plants and marine sources to partially or fully replace meat fats.Other studies have investigated alternative strategies, such as the use of plant starches and carrageenan, [4] olive oil as a fat replacer, [5] and formulations involving unripe banana by-products and pre-emulsified sunflower oil. [6]Notably, structuring edible oils is a key approach in this domain.
One such method involves a gel-filled emulsion or water-biphasic system that utilizes biopolymers (protein and/or polysaccharides) as gelling agents. [7]In the current study, sesame seeds in powdered form, or sesame seed flour, are employed.In a previously published journal, chia seed flour was used as a fat replacer, indicating its ability to enhance the stability of emulsion gels without affecting the pH and texture of fresh chicken formulations. [8]Unlike chia seeds, sesame seeds do not form a mucilaginous gel when soaked in water, and mucilages are promising thickening agents due to their colloidal structure. [9]hile this may contribute to the stability of sausage formulations, there is currently no published research on the functional properties of sesame seeds and their inclusion in meat products, specifically as seeds rather than as oil extracts.Furthermore, the use of sesame seed powder in low-fat labneh resulted in an increase in fat, protein, and ash and a reduction in microbial counts. [10]n the current formulation, the inclusion of whey protein isolate (WPI) and guar gum (GG) represents an effective method for binding oil and water.GG, a well-known polysaccharide in the food industry, is recognized for its neutral taste and odorless characteristics, making it ideal for enhancing the gelling strength of WPI in this study.Moreover, GG serves as a dietary fiber supplement with associated health benefits, including cholesterol level reduction and a significant decrease in the risk of heart disease. [11]A study by Rather et al. [12] revealed that the use of GG as a fat replacer influenced the quality characteristics of mutton goshtaba, an Indian food product.Furthermore, incorporating whey protein into food products has been shown to elevate nutrient levels. [13]Its versatility is evident in a study on banana cakes, where there were no alterations in the physical, chemical, and sensory qualities of the final product. [14]Literature on emulsified meat batters prepared at various protein levels suggests that preheating WPI is an effective strategy for improving the texture of WPI in meat emulsions. [15]Furthermore, the combination of WPI and sodium dodecyl sulfate has demonstrated enhancements in water-holding capacity, emulsion stability, and textural properties in low-fat sausages. [16]The interaction between whey protein and GG has shown good biocompatibility. [17]A study involving whey protein/GG pre-load revealed a delay in gastric emptying and stimulation of insulin secretion in healthy older individuals. [18]Therefore, the primary objective of this study was to assess the physicochemical and microbiological characteristics of chicken sausages formulated with sesame seeds, WPI, and GG at different inclusion levels.

Raw materials
Fresh breast chicken cuts were sourced from a local supermarket (Bandar Seri Begawan, Brunei Darussalam).The meat, devoid of all connective tissue and visible fats, was frozen overnight and then minced using a meat mincer equipped with a 6 mm sieve (AIFA, Model MG-N528).For the formulation, we procured WPI containing 100% pure whey isolate (LushProtein, Singapore), a foodgrade commercial preparation of GG (Take It Global Sdn Bhd, Penang, Malaysia), pure soybean oil (Prince, Malaysia), and sesame seeds from a local store (Bandar Seri Begawan, Brunei Darussalam).

Preparation of chicken sausages
The ground meat, after thawing, was supplemented with various additives, including food emulsifiers.Manual homogenization of the meat occurred in a food processor (Panasonic, MK-5076).The homogenization process continued until the desired emulsion was achieved, for approximately 1 min, with the temperature maintained at 12°C-15°C, measured using a handheld thermometer (Thermo Scientific, Model 1222W11).The uncooked batter from each formulation was manually filled into food-grade plastic sausage casings, each with a 50 mm diameter, using a sausage stuffer.
Three levels of fats were investigated in this study: 2%, 5%, and 10%, with the addition of 28%, 25%, and 20% of water, respectively.The aim was to maintain a consistent fat-to-meat ratio in the sausages, adhering to the typical sausage composition of 30% fat and 70% meat.All formulations shared the same common ingredients: 70% raw chicken meat and 30% added fat and water.As part of the sausage recipe, salt, garlic powder, and mixed herbs were also added to the samples.The control (CON) chicken sausage consisted of 70% lean breast meat without any animal fats.For each treatment, 100 ± 1 g of chicken sausages were precisely measured and replicated three times.The formulation percentages for all ingredients are displayed in Table 1.

Emulsion preparation
The emulsion was prepared by emulsifying WPI with distilled water using a food blender (KENWOOD, BL220 series) for 2 min.Oil was then added and homogenized for 5 min.Subsequently, gums and sesame seed flours were added and homogenized for an additional 3 min until a thick consistency was reached.The emulsion gels were refrigerated before adding them to the chicken mixture to create the batter.
As shown in Table 1, nine different types of oil-in-water emulsion gels were formulated.The first three types of emulsion gels were prepared using a stabilizer system based on sesame seed flour (S) and WPI only.Meanwhile, three emulsion types were prepared with 50% WPI and 50% GG (E) and another three emulsion types consisted 70% WPI and 30% GG (GM).

Cooking method
Sausages were thawed overnight, weighed before thawing, and then cooked at 175°C in a conventional oven until the internal temperature reached 70°C for 3 min, measured using a digital kitchen thermometer (Toriox, Model 1060). [19]The cooked sausage samples were refrigerated (4°C ±1°C) and frozen (−8°C ±1°C) throughout the analysis period.

Cooking loss
After cooking in the oven, the cooked chicken sausage samples were cooled for 30 min.The samples were weighed before and after cooking.The weight difference (%) between uncooked and cooked samples determined the cooking loss (CL). [20]

Water activity
The water activity (A w ) of each sample was measured in triplicate using a A w meter (WA-60A, Amtast) on days 0, 7, 14, 21, and 60 of the storage period.

Peroxide values
The peroxide value (POV) of samples was determined following the method described by Hwang et al. [21] with slight modifications.Approximately 0.5 g of each sample was weighed and placed in an Erlenmeyer flask.After heating in a water bath at 60°C for 3 min to melt the fat, it was agitated with a 25 mL acetic acid: chloroform (3:2 v/v) mixture for 3 min.The resulting mixture was filtered through Whatman filter paper to eliminate meat particles, and 1 mL of saturated potassium iodide solution was added.After keeping the mixture in the dark for 10 min, 30 mL of distilled water and 1 mL of starch solution (1% w/v) as an indicator were added.The sample was then titrated with 0.01 N sodium thiosulfate solution.The POV was expressed in milliequivalents of active oxygen per kilogram of the sample (meq/kg of sausage) using the formula: where S indicates the volume of titration in mL, N indicates the normality of sodium thiosulfate solution (N = 0.01), and W indicates the sample weight in g.

Determination of pH
The pH of the sausage samples was measured according to the method described by Sam et al.. [22] A pH probe connected to a continuous monitor (HM-500 Hydromaster) was used.A 5 g sausage sample was weighed with 50 mL of distilled water and homogenized thoroughly using a laboratory mortar and pestle.The pH was recorded three times for each sample on days 1, 7, 14, 21, and 60 of the storage period.

Aerobic plate counts
Microbiological evaluation of the sausages was conducted on days 1, 15, and 60 of the storage period, following the method according to Dikici et al. [23] with modifications.A 10 g sample of each sausage was mixed with 90 mL of 0.1% buffered peptone water.The homogenate was serially diluted, and triplicate 1 mL inoculum was spread on the agar.The agar plates were incubated at 37°C for 48 h.Counts of sausage samples were averaged and expressed as log CFU/g.

Statistical analyses
A two-way analysis of variance (ANOVA) was performed to evaluate significant differences (p < .05) in the effect of sausage samples.Additionally, a one-way ANOVA was used to evaluate CL%.Tukey's honestly significant difference test was applied to identify significant differences between all formulations and storage times.A p-value of less than 0.05 was considered statistically significant.Data in the tables are presented as mean values and standard deviations.

Cooking loss
The CL of chicken sausages formulated with various non-meat replacers exhibited significant differences (p < .05),as shown in Table 2. Sausage sample E10 demonstrated the lowest overall CL, followed by GM10, in comparison to other samples, attributable to their water-binding properties.Notably, W2 sausages displayed the highest CL value, closely followed by E2 when compared to the CON.This implies that the addition of a fat replacer reduced cooking losses and increased water-holding capacity.Consistent with prior studies, CLs tend to decrease with the addition of xanthan, [24] and the lowest CL was observed in meat products containing 1% alginate. [25]However, the CL values in this study were higher than those reported in 50% phosphate-reduced frankfurters [26] and fat-reduced frankfurters formulated using unripe banana by-products. [6]ter activity Table 3 presents A w measurements of chicken sausages under different storage conditions.On day 0, a statistically significant difference (p < .05)was observed among all treatments for both storage conditions.A w levels were in the range of 0.91-0.94A w for refrigeration and 0.88-0.95A w for freezing.Notably, a study reported A w of fresh chicken meats as 0.98 A w and cooked chicken meats as 0.96 A w . [27]The A w values in this study were higher than those reported for chicken nuggets using natural antioxidants. [28]ble 2 The addition of sesame seeds and other polysaccharides significantly impacted (p < .05) the refrigerated storage period.On day 7 of storage, the addition of WPI and GG resulted in higher values than to the CON (p < .05).By day 14, values were significantly similar to those of the CON.This aligns with findings using GG in mutton goshtaba [12] and low-fat Kadaknath chicken patties. [29]The ability of the gum to retain more water may compensate for the water content in the formulation. [30] w for all treatments then decreased on day 21, with the CON sample exhibiting the lowest values of 0.76.However, A w values increased again on the final day of storage, potentially due to heightened microbial activity.A w values exceeding 0.97 are indicative of high perishability. [31]In this study, without a fermentation process, hygienic quality became a primary factor influencing sample values.
Nonetheless, low A w is desirable.All treatments showed a significant decrease (p < .05)throughout the storage period, with the lowest A w values observed on day 60 during freezer storage, particularly in the GM5 treatment.This suggests that the ideal conditions for the treated sausages lie within freezer storage.This aligns with the observation that sausages with low A w exhibit better microbial stability [32] and effectively prevent the growth of bacteria, yeast, or molds. [33]

Peroxide values
The POVs, indicative of basic oxidation in sausage samples, are illustrated in Figure 1.A gradual increase in POV was observed for all treatments throughout the storage period (p < .05),from day 1 through day 60, under both refrigerated and freezer conditions.
Under refrigerated conditions, the CON sausage exhibited the highest POV, increasing from 4.92 meq/kg to 7.03 meq/kg of sausage, reaching 8.08 meq/kg of sausage at the end of the storage period.This trend differed notably from all other treatments.Similarly, in the freezer storage condition, the CON sample also had the highest POV, indicating the most significant lipid oxidation, aligning with previous reports. [22,34]Among all low-fat chicken sausages under refrigerated conditions, GM10 exhibited the lowest initial POV at 1.94 meq/kg of sausage, followed closely by E5 and W5 with POV values of 3.13 meq/kg of sausage and 3.18 meq/kg of sausage, respectively.
The initial POVs of chicken sausages containing varying levels of WPI and GG exhibited no significant differences (p < .05)under freezer conditions.A trend of declining POVs on day 7, followed by an increase on day 14 was noted, particularly in half of the treatments under freezer conditions.This suggested that after the induction rate, the decomposition rate of hydroperoxides surpassed the production rate. [35]Ultimately, POVs of all treatments remained below the recommended range of 20 meq/kg to 40 meq/kg, associated with a notable rancid taste. [36,37]

Determination of pH
Table 4 presents the effect of WPI and GG on the pH of chicken sausages under different storage conditions.The presence of WPI and GG in the formulations significantly affected the pH values (p < .05)among treatments throughout the storage period under refrigerated conditions.
A statistically significant difference (p < .05)among all samples was observed on day 1 under refrigerated (4°C ±1°C) conditions, with different inclusion levels and combinations of polysaccharides.On day 2, the pH values of all samples significantly (p < .05)decreased.pH values during the first day of storage until day 14 were lower than those reported in a study that used xanthan, guar, carrageenan, and locust bean gum in meatballs, ranging between 6.7 and 6.9 for cooked meatballs. [38]urthermore, the pH values significantly increased (p < .05) on day 21 with the addition of WPI and GG.The increased pH on day 21 during the refrigeration could be attributed to the growth of aerobic bacteria and an increase in microbial metabolites.Gill CO [39] reported that bacteria, on the depletion of stored glucose, utilize amino acids during protein breakdown.The consequent byproduct of amino acid degradation, ammonia accumulation, results in an increase in the pH value.This inference corresponds with the results of the aerobic plate counts reported in this study, indicating increased aerobic plate counts throughout the refrigerated storage period.At the end of the refrigerated storage period, significant differences (p < .05)were observed among the pH values of the treated sausages.These values slightly decreased compared to those recorded on day 21 during storage.Notably, only the E2 sample sausages had a minor, yet significantly higher (p < .05),pH value than the CON sample sausage.In contrast to a previous study, [40] the addition of whey protein concentrates (WPCs) and gums resulted in no significant effect on pH throughout the storage period, similar to the addition of carrageenan and pectin to frankfurters. [41]n addition, overall pH values on days 21 and 60 were higher in refrigerated storage than in freezer storage.This increase in pH may be attributed to metabolite accumulation, which tends to be higher under refrigerated storage conditions due to the growth of microorganisms in the sausage.A similar finding of increased pH values has been reported in chicken meatballs. [42]amples stored in a freezer (−8°C ±1°C) throughout the storage period also exhibited a significant increase (p < .05)(Table 4).All treatments formulated with WPI and GG exhibited significantly lower pH values than the CON on day 1 of freezer storage.Treatments with only WPI exhibited values that were similar to the CON.The decrease in pH values with the addition of GG can likely be attributed to the high temperatures during the cooking process, which can reduce the stability of GG. [43] However, on days 2 and 14 during storage, the addition of WPI and GG resulted in no significant effects (p > .05) on the pH of chicken sausages compared to the CON.Moreover, the final increase in pH values on days 14 and 21 was more pronounced with the addition of GG.This increase in pH on day 21 was in the range of 5.5 to > 6, potentially resulting from imidazolium exposure, a base in histidine amino acids, during protein denaturation. [44]By the end of the 60-day storage period, the pH values further declined but exhibited an insignificant (p > .05)deviation compared to the CON.This declining pH trend is consistent with the findings of WPC in buffalo meat, wherein a decline in the pH value of emulsion sausage samples was reported. [45]

Aerobic plate counts
The alterations in microbiological counts over a 60-day freezer storage period (−8°C ± 1°C) are detailed in Table 5.Initially, the aerobic count ranged from 4.07 CFU/g in E5 to 4.77 CFU/g in E10 and GM2.No significant differences were observed between CON and treated sausage samples at the start of the storage, consistent with a study investigating the use of plant starches and carrageenan during the initial 2 days of storage. [4]As the storage period advanced, a noticeable escalation in total bacterial counts occurred.On the day of storage, the total aerobic counts of treated sausages were significantly higher (p < .05)than that of the CON sausages (4.15 CFU/g).This increase can be ascribed to elevated A w and additional water in the formulation compared to the CON.A similar effect was also noted in low-fat frankfurters with the presence of carrageenan. [41]owever, no significant impact of WPI and GG addition on microbial activity in chicken sausages was observed between treatments on days 15 and 60.This suggests that the treatments might induce a delay in microbial proliferation during the freezing period.Importantly, the products remained microbiologically safe during the 60-day storage, with total plate count values consistently below the incipient spoilage level of 5.30 log CFU/g, [46] meeting the standards set by food authorities. [47]All treatments remained within permissible limits.
When comparing the microbiological quality of chicken sausages stored in refrigerated conditions (4°C ± 1°C), acceptable quality was attained only after the first day of storage.Figure 2  comparison of chicken sausages during the initial day of both storage conditions.Treated samples under refrigerated storage conditions exhibited an increasing trend in microorganisms (Figure 3) with a statistically significant (p < .05)increase in aerobic plate count, ranging from 5.33 log CFU/g to 6.21 log CFU/g over the 60-day period.Similar increases in total aerobic count with prolonged storage have been previously reported. [34,48,49]This increase may be attributed to the water content present in the formulation, providing an environment conducive to bacterial growth. [50]This observation is further supported by the increase in A w values in this study.It is crucial to note that the total plate count of refrigerated chicken sausages treatments exceeded the acceptable microbiological limit of 5.3 log CFU/g.None of the treatments can comprehensively explain the significance of the changes in the formulation during the 60-day shelf life.Therefore, further investigations are required to evaluate the effects of extended freezing storage periods on microbial activity, including yeasts and molds.Such studies are essential to determine a decisive factor for product acceptability.

Conclusion
This study highlights the efficacy of incorporating emulsion gels as a fat replacer in chicken sausages, yielding higher cooking values compared to previous research.The observed POV in this study remains within acceptable ranges for fatty foods, with no significant impact from the additions of WPI and GG.Significant differences (p < .05) in pH values were noted throughout both the storage period and across various treatments.Crucially, experimental sausages were acceptable for consumption up to 60 days when frozen (−8°C ± 1°C), supported by lower A w values with the addition of GG under the same storage conditions.However, sausages were deemed unacceptable under refrigeration (4°C ± 1°C) conditions starting day 15.Future studies must explore antioxidant activity and other microbiological aspects to better understand the potential of this fat replacer.The findings of our study suggest that incorporating sesame seeds, WPI, and GG holds promise for enhancing the nutritional value of chicken sausages.

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

Figure 1 .
Figure 1.Peroxide values of control samples and chicken sausages treated with whey protein isolate (WPI) and guar gum (GG) stored at 4°C and − 8°C throughout storage period.(a) Control with 100% WPI under refrigerated conditions.(b) Control with 50% WPI and GG under refrigerated conditions.(c) Control with 70% WPI and 30% GG under refrigerated conditions.(d) Control with 100% WPI under freezer conditions.(e) Control with 50% WPI and GG under freezer conditions.(f) Control with 70% WPI and 30% GG under freezer conditions.Different letters above the standard error bars indicate significant differences between the storage periods (p < .05).

Figure 2 .Figure 3 .
Figure2.Aerobic plate counts of the control and experimental chicken sausage samples with the addition of sesame seeds, WPI, and GG under two storage conditions.a-c indicates with significant differences (p < .05)among all treatment and control samples.

.
Effects of the treatment of sesame seeds, WPI, and GG on the cooking loss of lowfat chicken sausages.

Table 3 .
Water activity (A w ) of sausage samples under refrigerated and freezing storage conditions for 21 days.
aDDifferent subscripts in the same column and rows indicate the significantly different average values among samples (p < .05)(mean ± SE). a-h indicates significant differences (p < .05)among treatments.A-E indicates significant differences (p < .05)among

Table 4 .
The pH of sausages under different storage conditions.
aCDifferent subscripts in the same column and rows indicate the significantly different average values among samples (p < .05)(mean ± SE). a-f indicates significant differences (p < .05)among treatments.A-F indicates significant differences (p < .05)among storage periods.

table 5 .
Microbiological quality of chicken sausages under freezer storage conditions for 60 days.
aADifferent subscripts in the same column and rows indicate the significantly different average values among samples (p < .05)(mean ± SE). a-h indicates significant differences (p < .05)among treatments.A-C indicates significant differences (p < .05)among storage periods.