Evaluation of Hesperidin on Sausages Stuffed in a New Modified Casing during Long-Term Storage—A Preliminary Study

Abstract

The effects of hesperidin and different casings on pH, moisture content, water-holding capacity, and total viable count (TVC) of sausages stored for 171 days were evaluated by principal component analysis (PCA) and discriminant analysis (DA). Sausages stuffed in a modified casing with treatment B (soy lecithin concentration: 1:30, soy oil concentration: 2.5%, lactic acid concentration: 21 mL/kg NaCl [solid], treated time: 90 min) had a significantly lower pH value (6.89 ± 0.01) at d 31 but higher (6.55 ± 0.03) than that of control casings at d 171 (p < 0.05). Hesperidin plays an important role in antimicrobial property that renders the sausages with modified casing by treatment A (soy lecithin concentration: 1:27.5, soy oil concentration: 1.25%, lactic acid concentration: 19.5 mL/kg NaCl [solid], treated time: 75 min) stable (p > 0.05), with the final TVC of 5.03 ± 0.10 log cfu/g. According to the PCA results, water-holding capacity has a positive correlation to pH. Moisture content was the best discriminator for differentiating sausages with control and modified casings, whilst pH was able to discriminate sausages stored after 138 days from other days.

1. Introduction

Citrus fruit peels contain a large amount of useful and bioactive compounds such as flavonoids and phenolic acids, which play important roles in keeping people healthy because several flavonoids cannot be synthesized biologically [1]. Hesperidin, as one of the most important flavonoids from the Citrus species, exhibits various biological properties such as antioxidant, anticancer, and antimicrobial properties, and so on [2]. Recently, it has been found to potentially prevent coronaviruses, which may be due to its better binding affinity to the main protease of COVID-19 [3]. The other theoretical site was claimed to require lower energy to bind hesperidin with SARS-CoV-2, which avoids the virus binding with the host cell [4]. On the other hand, approximately 1000 tons of orange waste was generated in Shizuoka prefecture in Japan with around 400 thousand tons of consumption of fruit juice [5]. Therefore, it is highly meaningful to extract bioactive compounds from waste orange peels to reduce the environmental problem and maximize reuse [6], potentially maintaining sustainability development.

As an important gastronomic and nutritional heritage, sausages exert an important impact on people’s daily life [7,8]. Natural casings, with their special bite and unique flavor, are superior to other artificial casings [8]. Nevertheless, the burst incidence generated during handling and process is the main hindrance to its widespread. As a result, food-grade surfactant solutions associated with slush salt with lactic acid were employed to improve the quality of the casings [9]. Casings after modification were reported to be more porous and even fat was embedded into the casings [10]. Although this structure could efficiently reduce the burst incidence during sausage stuffing or other procedures, the porous structure may increase the risks of oxidation or microorganism invasion. It is thus necessary to add food preservation to prolong the shelf life of the sausage without compromising the pressure resistance of the modified casings. As hesperidin possesses antioxidant and antimicrobial properties, it is worthy to investigate how sausages produced by the unique modified casings respond to the addition of hesperidin.

As a useful tool to interpret large datasets by forming fewer composite variables, principal component analysis (PCA) has been widely applied to analyze the volatile compounds of Harbin red sausages smoked with different woodchips [10], quality of cooked sausages with different types of casings [11], volatile organic compounds of Portuguese Painho de Porco Preto fermented sausages [12], texture profile of sausage added of pea protein isolate [13], and so on. Discriminant analysis (DA) is also a useful tool to determine possible categorical dependent variables based on the selected variables [14,15]. However, scarce information has been reported on the effects of hesperidin on the quality and microbial attributes of sausages stuffed in modified casings treated with different concentrations of surfactant solution and lactic acid. The innovative points of the current study are as follows:

• The interactive effects of hesperidin and modified casing on the quality parameters of sausages during long-term storage are elucidated, which provides useful information to protect sausages with different modified casings and potentially extend their shelf lives.
• The relationships between the measured parameters and individuals such as different casing treatments added with hesperidin and storage days are established for the first time by PCA and DA.

The objective of this work is thus to investigate the effects of hesperidin on sausages with different casings during long-term storage using PCA and DA.

2. Materials and Methods

2.1. Sausage Preparation

The visible fat (Watasei Co., Ltd., Utsunomiya City, Tochigi Prefecture, Japan) was removed from fresh lean pork purchased at Passport Co., Ltd. Fukaya Meat Center, Saitama Prefecture, Japan. A total of 4238 g of lean pork and 2511 g of fat were sterilely cut into small pieces and mixed well with the following ingredients: Chinese white wine (845 g; ethanol content: 52%, v/v), black pepper (87 g), coriander powder (44 g), spicy pepper (50 g), salt (235 g), sugar (129 g), monosodium glutamate (14 g), and standard hesperidin powder (27 g, Tokyo Chemical Industry Co. LTD, Tokyo, Japan). After being cured for 1 day at 4 °C, the meat with ingredients was stuffed by a stuffing machine (STX-4000-TB2-PD-BL, Electric Meat Grinder & Sausage Stuffer, STX International, Lincoln, NE, USA) using desalted normal hog casings (as a control) and hog casing modified by the following treatments:

The ingredients of the surfactant solution were dissolved using distilled water via a magnetic agitation with 325 rpm at 60 °C. The casing was put into a surfactant solution after it cooled to 25 °C with treated time. Subsequently, the casings were taken out without rising and mixed with slush salt with lactic acid with another treated time. The composition modified solution and treatment time for treatments A and B can be found in

Table 1. Treatment of modified casings.

Treatments	Surfactant Solution		Treated Time in Surfactant Solution (min)	Lactic Acid Concentration (mL/kg NaCl)	Treated Time in Lactic Acid (min)
	Soy Lecithin 1	Soy Oil (%, w/w)
A	1:27.5	1.25	75	19.5	75
B	1:30	2.5	90	21	90

¹ Soy lecithin: distilled water (w/w).

. The sausages were hung and dried in a sterilized oven at 65 °C for 24 h and aged at 27 °C for another 48 h. The detailed sausage and casing-modification processing can be found in Feng et al. (2014) [9]. The sausage cuts were vacuum-packaged and stored at 4 °C for the following post analysis. The main procedures are shown in Figure 1.

2.2. Physicochemical Analyses

According to the method of Zdolec et al. (2008) [16], ten grams of sausages were homogenized with 90 mL distilled water for 1 min and measured using a digital pH meter.

As for moisture analysis, 5 g of minced samples were dried in an oven at 105 ± 1 °C until constant weight. The moisture content of the sample was calculated as follows:

$$\mathrm{Moisture} \mathrm{content} (\%)=\frac{W_b-W_a}{W_b} \text{×} 100\%$$

(1)

where W_b and W_a are the weights before and after drying.

Regarding the water-holding capacity (WHC), the sausages’ weights before and after centrifuging (4 °C, 9000× g, 10 min) were calculated as the WHC [11].

2.3. Total Viable Count (TVC) Enumeration

A laboratory stomacher (E-Mix Primo, ASONE interscience Co., Inc., Osaka, Japan) was employed to sterilely homogenize ten grams of sausages (with casings) using 90 mL of 0.85% saline for 2 min. After tenfold serial dilution using the 0.85% saline, 0.1 mL of diluted sample was spread onto CicaMedia Standard plate-count agar (Kanto Chemical Co., Inc., Tokyo, Japan, pH: 7.0 ± 0.2), followed by incubating at 37 °C for 48 h.

All the experiments were conducted in triplicate. Physicochemical analyses were conducted on days 1, 10, 12, 18, 31, 43, 127, 138, 143, 166, and 171, whilst TVC enumeration was carried out on days 1, 9, 11, 18, 31, 43, 138, 143, 166, and 171.

2.4. Statistical Analysis

PCA was utilized to categorize quality attributes and sausages with different casings. The differences among the different treated sausages and storage days with regard to physicochemical and TVC parameters were analyzed by analysis of variance (ANOVA, p < 0.05). All the analyses were implemented by Minitab (21.1 Minitab Statistical Software, Kozo Keikaku Engineering Inc., Tokyo, Japan). A canonical discriminant analysis was employed to estimate the sausage storage days and different treatments according to pH, water-holding capacity, moisture content, and the TVC via the software SPSS (Statistics 28.0.1.1 (15), IBM, Armonk, NY, USA).

3. Results and Discussion

3.1. Effects of Different Treatments on PH of Sausages

The average pH value of sausages stuffed in casing modified by treatment B (6.89 ± 0.01) was significantly lower than that of control casings (7.04 ± 0.01) at d 31 (p < 0.05) but significantly higher than that of control casings at d 171 (6.55 ± 0.03 Vs. 6.40 ± 0.04; p < 0.05) (

Table 2. pH, WHC, and moisture-content comparison for sausages with different treatments during 171 days of cold storage (4 °C).

Parameters	S	d 1	d 10	d 12	d 18	d 31	d 43	d 127	d 138	d 143	d 166	d 171
pH	C	6.89 ± 0.01 BCa	6.98 ± 0.03 ABa	6.96 ± 0.01 ABa	6.91 ± 0.01 BCab	7.04 ± 0.01 Aa	6.95 ± 0.02ABa	6.89 ± 0.01 BCa	6.83 ± 0.01 Ca	6.31 ± 0.06 Db	6.37 ± 0.07 Db	6.40 ± 0.04 Db
	A	6.94 ± 0.01 Ab	6.86 ± 0.12 ABa	6.90 ± 0.01 Ab	6.87 ± 0.03 Ab	6.97 ± 0.03 Ab	6.94 ± 0.01Aab	6.74 ± 0.02 Bb	6.74 ± 0.03 Bb	6.34 ± 0.03 Dab	6.43 ± 0.02 CDab	6.49 ± 0.03 Ca
	B	6.98 ± 0.01 Ac	6.91 ± 0.08 ABa	7.00 ± 0.01 Ac	6.95 ± 0.01 ABa	6.89 ± 0.01 Bc	6.91 ± 0.01ABb	6.77 ± 0.02 Cb	6.73 ± 0.02 Cb	6.43 ± 0.02 Ea	6.52 ± 0.02 Da	6.55 ± 0.03 Da
WHC (%)	C	99.35 ± 0.06 Aa	97.95 ± 0.91 Aa	98.88 ± 0.19 Aab	97.61 ± 0.25 Ab	98.56 ± 0.31 Aa	98.52 ± 0.27Aa	92.33± 1.12 Ba	89.49 ± 1.15 BCa	90.94 ± 1.46 BCa	88.44 ± 1.25 Ca	89.78 ± 2.25 BCa
	A	97.87 ± 0.78 Ab	99.08 ± 0.38 Aa	98.90 ± 0.27 Aa	98.87 ± 0.07 Aa	99.19 ± 0.24 Aa	97.69 ± 0.31Aa	89.68± 0.54 Bb	86.40 ± 2.66 Ba	87.95 ± 0.52 Bb	91.83 ± 8.50 ABa	85.84 ± 0.97 Bb
	B	98.82 ± 0.39 Aab	98.68 ± 0.27 Aa	98.32 ± 0.22 Ab	98.93 ± 0.27 Aa	98.95 ± 0.19 Aa	96.93 ± 2.48 Aa	88.38 ± 0.99 Bb	87.49 ± 1.90 BCa	84.14 ± 0.93 Dc	85.87 ± 0.41 BCDa	85.06 ± 1.13 CDb
MC (%)	C	28.08 ± 4.33 DEa	40.63 ± 3.59 ABCa	37.45 ± 2.11 ABCa	35.79 ± 2.23 BCDa	45.04 ± 4.14 Aa	41.91 ± 4.20 ABCa	38.28 ± 2.02 ABCa	40.55 ± 3.99 ABCa	44.09 ± 2.39 ABa	25.14 ± 2.20 Ea	34.48 ± 0.48 CDa
	A	22.23 ± 3.50 BCa	21.63 ± 3.34 BCb	19.72 ± 2.32 BCb	25.93 ± 2.24 ABb	24.33 ± 1.54 ABCb	25.02 ± 1.32 ABb	24.76 ± 1.26 ABCa	21.72 ± 1.70 BCb	29.03 ± 1.93 Ab	18.53 ± 2.60 Cb	22.19 ± 0.78 BCb
	B	24.52 ± 4.28 Aa	28.41 ± 2.62 Ab	26.03 ± 2.65 Ac	21.08 ± 0.62 Ac	27.78 ± 3.26 Ab	24.32 ± 2.49 Ab	27.87 ± 9.79 Aa	21.53 ± 2.54 Ab	21.31 ± 3.52 Ac	22.81 ± 0.67 Aab	25.85 ± 0.51 Ac

Note: Sausages stuffed by control hog casing: C; modified casings with treatment A: A; modified casings with treatment B: B; WHC: water-holding capacity; MC: moisture content; S: sample. Different lower letters within a column are significantly different (p < 0.05). Different upper letters within a row are significantly different (p < 0.05).

). A significantly lower pH value for a sample of treatment B (6.37 ± 0.00 Vs. 6.84 ± 0.00 for the control sample) at d 36 was also observed (p < 0.05) [17], which may be due to a more porous structure produced under treatment B. In regard to the storage days, the mean pH value of sausage with control casing increased from 6.89 ± 0.01 to 7.04 ± 0.01 on d 31 and finally decreased to 6.40 ± 0.04. (p < 0.05). The effects of different concentrations of orange fiber powder (mixture of albedo, flavedo, and pulp) on the quality of dry-cured sausages during the 28-day dry-curing time (15 ± 1 °C, relative humidity: 75%) were investigated [18]. The pH value of the control sample was reported to decrease during the first week and then increase back to a similar value after 4 weeks. A similar tendency was presented in samples added with orange fiber irrespective of the concentration. The higher concentration of orange fiber used, the lower the pH value was [17]. The hesperidin contents (0.062 ± 0.009 mg/g for sausages with 20 g/kg orange fiber) showed an insignificant difference with regard to the processing time (p > 0.05) [17]. However, others reported no significant differences observed regarding citrus fiber concentration or storage time (28 days) on pH level (p > 0.05) [18]. Viuda-Martos et al. (2010) studied the pH value evolution for bologna sausage after adding orange dietary fiber (ODF), oregano essential oil (OEO), and different packaging conditions [19]. There was a slight decrease from 6.2 to 6.1 for samples with ODF + OEO at d 18 (p > 0.05), and an increase again, although not significant at a 5% level (p > 0.05). However, the hesperidin levels dropped significantly from 416.5 ± 1.6 μg/g (d 0) to 411.3 ± 0.1 μg/g (d 24) (p < 0.05) for vacuum-packaged samples [19].

Different from the aforementioned study, pure hesperidin (purity > 95%) and longer storage days (at 4 °C) were applied. The pH values of samples stuffed in casings with treatment A and B gently decreased at d 10 but rose at d 12, although not to a statistical significance (p > 0.05). They finally dropped to 6.49 ± 0.03 and 6.55 ± 0.03 at d 171, respectively, which may be attributed to the glycogen breakdown and consequent generation of lactic acid, leading to the decrease in pH value [20]. Water extracts from psyllium seeds, locust ben seeds, and orange albedo as well as pure pectin were added to beef sausages stuffed in the nature casings [21]. The pH values for all the samples decreased; only the samples with psyllium extracts (the highest hesperidin content 442.4 mg/100 g) had a comparably higher pH value at 6 months (approx. 180 days) under frozen (−18 °C) [21].

3.2. Effects of Different Treatments on Water-Holding Capacity of Sausages

As elaborated in Table 2, sausages with control hog casings possessed a significantly higher WHC (89.78 ± 2.25%) than those with modified casing (85.84 ± 0.97 for treatment A and 85.06 ± 1.13 for treatment B) on the final day (d 171, p < 0.05). This may be due to the porous structure of the modified casing, although hesperidin has been added as an ingredient. It did not seem to improve the WHC of sausages in the current study, although hesperidin was expected to hinder protein oxidation. As an important parameter to evaluate the ability of meat to hold water during exterior mechanical force [22,23], it plays an important role in consumers’ mouthfeel of meat products. It can be observed that the WHC value significantly decreased on the final day irrespective of the treatments (p < 0.05). This may be due to the protein oxidation that destroys the structure of proteins and affects protein properties [24]. The effects of litchi (Litchi chinensis Sonn.) flower powder water extracts (LFPWE) on the water-holding capacity of emulsified pork meatballs were investigated; 0.15 ± 0.01 mg/100 mL of neohesperidin was detected from ascorbic acid in 1% (w/v) LFPWE and the WHC of cooked meat products worsened with a time of 4 weeks of storage (−20 °C) [24]. The authors attribute this observation to protein oxidation. The lower contents of myofibrillar proteins such as myosin and actin were observed in the control group, leading to a lower firmness of emulsified pork meatballs and poor WHC value [24]. It can be observed that the WHC value significantly decreased on d 127 irrespective of the treatments (p < 0.05), indicating the occurrence of protein oxidation. Protein denaturation and loss of protein solubility may also contribute to the decreased WHC values [21].

3.3. Effects of Different Treatments on the Moisture Content of Sausages

The moisture of the control sample did not show a significant difference compared with samples treated by A and B at the beginning (i.e., d 1) (p > 0.05) (Table 2). However, the moisture content of the control sample (40.55 ± 3.99%) was significantly higher than that of modified casing samples with treatments A (21.72 ± 1.70%) and B (21.53 ± 2.54%) at d 138 (p < 0.05) (Table 2). It was reported that the water distribution in sausage is primarily immobilized water [25]. At the beginning of the storage, it is a suitable environment for Gram-positive bacteria growth. The lower moisture and pH refrained the total bacterial growth [25]. The high difference and jumps and downs of moisture content in control treatment can be probably explained by the equilibrium between mass-transfer processes, diffusion, and evaporation between the free surface of sausage and water diffused towards the surrounding [26]. As the porous structure of the modified casing, it will render moisture content to easily achieve equilibrium and thus give the moisture content relevant stability. Concerning the whole 171 days of storage, samples stuffed in casings with treatment B showed no significant difference (p > 0.05) and were comparably stable to that of treatment A, indicating that different concentrations of modified solution affected the moisture-content stability.

3.4. Effects of Different Treatments on the Total Viable Count of Sausages

The TVC elaborates on the quantity of the dominant microorganism and is recognized as an essential parameter to evaluate the shelf life of the foodstuffs. For samples in the control group and treatment A, there were no significant differences between d 1 and d 171 (p > 0.05) (

Table 3. Total viable count comparison for sausages with different treatments during 171 days of cold storage (4 °C).

Samples	d 1	d 9	d 11	d 18	d 31	d 43	d 138	d 143	d 166	d 171
Control casing (log cfu/g)	5.78 ± 0.56 ABCa	6.56 ± 0.29 Aa	4.73 ± 0.00 Ca	5.18 ± 0.11 BCa	5.81 ± 0.67 ABCa	4.95 ± 0.01 BCa	6.30 ± 0.49 ABa	4.65 ± 0.00 Ca	5.50 ± 0.01 ABCa	5.51 ± 0.11 ABCa
Treatment A (log cfu/g)	5.19 ± 0.12 Aab	5.45 ± 0.27 Ab	5.43 ± 0.68 Aa	4.86 ± 0.00 Aa	4.95 ± 0.00 Aa	4.80 ± 0.21 Aa	5.91 ± 0.06 Aa	5.99 ± 0.87 Aa	5.40 ± 0.00 Aa	5.03 ± 0.10 Aa
Treatment B (log cfu/g)	4.34 ± 0.12 Bb	5.97 ± 0.47 ABab	4.91 ± 0.07 Ba	4.99 ± 0.37 Ba	5.08 ± 0.25 Ba	4.73 ± 0.00 Ba	6.00 ± 0.00 ABa	5.68 ± 0.01 ABa	5.65 ± 0.43 ABa	7.35 ± 1.10 Aa

Note: Different lower letters within a column are significantly different (p < 0.05). Different upper letters within a row are significantly different (p < 0.05).

). The total viable counts of control and modified casings (treatment A) without adding hesperidin at d 43 were reported as 8.62 ± 0.69 log cfu/g and 7.25 ± 0.70 log cfu/g, respectively [27]. There were obvious deductions for those samples by adding hesperidin: 4.95 ± 0.01 log cfu/g for control and 4.80 ± 0.21 log cfu/g for samples with treatment A (Table 3), indicating the antimicrobial property of hesperidin during long-term storage. Osheba et al. (2013) studied the effects of extracts from psyllium, locust bean, and orange peel albedo on the total bacterial count of beef sausages after 6 months frozen storage [21]. The total bacterial count of beef sausages with extracts from psyllium (4.31 × 10⁵ cfu/g = 5.63 log cfu/g) was the lowest, compared to that added with extracts from locust bean (5.85 × 10⁵ cfu/g = 5.77 log cfu/g) and orange peel albedo (7.18 × 10⁵ cfu/g = 5.86 log cfu/g). The hesperidin content was reported to be 442.40 mg/100 g for psyllium, 265.18 mg/100 g for locust bean, and 65.14 mg/100 g for orange peel albedo, respectively [21]. Several mechanisms such as interference with bacterial DNA synthesis, bacterial movement, cytoplasmic membrane permeability, and bacterial metalloenzymes inhibitions have been utilized to explain its antibacterial activities [2,28,29].

Regarding samples in the modified casing with treatment B, Table 3 displays that the TVC count increased significantly on the last day (7.35 ± 1.10 log cfu/g) in comparison with d1 (4.34 ± 0.12 log cfu/g) (p < 0.05). This may be due to the different hole sizes generated by different concentrations of the modified solutions. According to the light-microscopy images of cross sections of sausages stuffed in modified casings with treatments A and B, the fat was embedded inside the modified casing with treatment B after immersion vacuum cooling, whilst this phenomenon can be only observed via a transmission electron microscope in the case of treatment A casing [9]. This indicates that the hole size of casings modified by treatment A was smaller than that of treatment B. Although hesperidin possesses an antimicrobial property, this ability will be weakened if the larger porous structure renders the microorganisms easily invadable. The best combination of modified solution and hesperidin concentration merits further investigation.

3.5. Principal Component Analysis and Discriminant Analysis

Table 4. The variation source from the first two main components.

Parameters	Component
	1	2
Eigenvalue	3.05	1.02
Proportion (%)	61.10	20.40
Cumulative (%)	61.10	81.50
pH	0.51	0.02
Water-holding capacity (%)	0.56	0.06
Moisture content (%)	0.10	0.94
Total viable count (log cfu/g)	−0.34	0.34
Storage days	−0.56	0.04

displays the first two principal components (PC) that can explain 81.50% of the whole attributes. Accordingly, PC 1 explained 61.10% of the total variation in all attributes while PC 2 explained 20.4%. Figure 2b depicts the water-holding capacity has a positive correlation to pH. Puolanne et al. (2001) studied the combined effects of NaCl and raw meat pH on water-holding capacity in cooked sausage with and without adding phosphate [30]. With the 2.5% NaCl, the maximum WHC value obtained and the pH had the largest effect. It was concluded that high pH is related to the WHC and firmness [2]. Regarding the current study, 235 g (i.e., 2.87%, w/w) of salt was added to the sausage; a higher pH value resulted in a higher WHC value. Salt exacts three important impacts on meat proteins: protein–protein (meat-binding), protein–water (water-holding), and protein–fat (fat-binding) [31]. It not only affects the dissolved protein but also insoluble meat fibers and connective tissue composed of minced meat batter (i.e., sausage filling) [31]. It was explained that chloride ions penetrate myofilaments, resulting in swelling and generating the filaments, which attributes to the increase in water-holding capacity [32]. This is consistent with the observation where the best WHC value was achieved with the highest pH value reached during 6 months of frozen storage of beef sausage [21].

Moisture content, water-holding capacity, and pH were located on the opposite side of storage days and total viable count, indicating a negative correlation. It is well-known that the lower pH and moisture content will hinder the growth of microorganisms, leading to the lower value of TVC.

According to discriminant analysis, two discriminant functions were obtained to separate sausages with different treatments, with the correct classification of 66%. The two functions were as follows:

Function 1 = 0.94 [moisture content] + 0.12 [WHC] − 0.03 [TVC] − 0.03 [pH]

(2)

Function 2 = 0.29 [moisture content] − 0.08 [WHC] + 0.72 [TVC] + 0.36 [pH]

(3)

As displayed in

Table 5. Statistics of the canonical discriminant functions were calculated from 4 variables.

Separation	Canonical Function	Eigenvalue	Variance (%)	Canonical Correlation	Wilks’ λ	p-Value
Treatments	Function 1	1.75	96.6	0.80	0.34	p < 0.01
	Function 2	0.06	3.4	0.24	0.94	p > 0.05
Storage days	Function 1	56.00	97.9	0.99	0.01	p < 0.01
	Function 2	0.89	1.6	0.69	0.39	p < 0.01
	Function 3	0.22	0.4	0.42	0.74	p > 0.05
	Function 4	0.12	0.2	0.32	0.90	p > 0.05

, Function 1 explains 96.6% of the total variance and possesses a higher canonical correlation (0.80) at a 1% significant level, which indicates that Function 1 had a comparably higher reliability. A pronounced discriminatory ability is shown due to its low Wilks’ lambda value.

The variable with the highest discriminant power (highest absolute coefficient value) in Function 1 was moisture content, followed by water-holding capacity (Equation (2)). As illustrated in Figure 3a, sausages with control casings can be classified by moisture content in groups of sausages with modified casings. This is consistent with the observation that the moisture content was higher than modified casings irrespective of the storage days (Table 1).

Regarding samples with different storage days, four canonical discriminant functions were established with 54.7% correct classification. The first two functions, with a significant level of 1%, are displayed as follows:

Function 1 = 0.42 [WHC] + 0.58 [pH] + 0.01 [moisture content] − 0.07 [TVC]

(4)

Function 2 = −0.83 [WHC] + 0.82 [pH] − 0.04 [moisture content] + 0.46 [TVC]

(5)

Function 1 explained 97.9% of the total variance (Table 5) and pH was the most powerful discriminant variable to separate sausages stored after 138 days from other days. Sausages stored for 143 days were mainly located in the negative part of Function 2, whilst samples with 171 and 138 days’ storage were located in the positive part. Water-holding capacity had the highest discriminant power by Equation (5), indicating that WHC changed greatly in those days.

This is the preliminary study of the effects of hesperidin on sausages stuffed in this unique modified casing; future research on protein oxidation, chemical composition, texture profile, sensory analysis, and lipid oxidation will be conducted in the near future.

4. Conclusions

This research investigated the effects of adding hesperidin on quality and microbial attributes during 171 days of cold-room storage. Sausages with hesperidin indeed refrained from the increase in total viable count, and no significant differences were found in sausages stuffed in the modified casing with treatment A during storage. Discriminant analysis can distinguish the sausages with differently treated casings based on the quality attributes and TVC with 66% correct classification. Sausages with treatment B had a significantly lower pH (6.89 ± 0.01) at d 31 but higher (6.55 ± 0.03) than that of control casings at d 171 (p < 0.05). Water-holding capacity has a positive correlation to pH, and moisture content was the best discriminator for distinguishing sausages with control and modified casings. Although a few basic quality attributes have been carried out in this preliminary study, it offers useful information on how moisture, WHC, pH, and TVC respond to hesperidin. Moreover, the relationship between quality attributes and bacterial growth during long-term storage of sausages with different casings adding hesperidin has been elucidated in this preliminary study, which could be of practical application to sausages using modified casing being commercially utilized. Deeper analyses such as oxidation, shelf life, textural, and specific microbial analysis (lactic acid bacteria, Enterobacteriaceae, Pseudomonads, and so on) need to be confirmed in future work.