Effect of garlic oil and chitosan coating on preservation of Labeo rohita meat fillets in refrigerated conditions

Abstract

Fish is an important food source containing high quality proteins, vitamins and minerals. The spoilage of fish meat causes a huge economic loss at local and commercial level that can be minimized with efficient preservation. The demand of natural organic preservatives is increasing in recent years due to the hazardous effects of synthetic preservatives. Among the natural preservatives, essential oils in combination with chitosan have been proved a good edible meat preservative candidate. Consequently, we carried out our work to evaluate the preservative effect of garlic oil and chitosan, both solo and in combination, on refrigerated Rohu (Labeo rohita) meat by following two-factor factorial completely random design (CRD) experimental design. Garlic oil (0 & 1%) and chitosan (0 & 1%) in 2 × 2 interaction was applied on refrigerated fillets of Rohu meat. The methodology involved weekly evaluation of proximate composition, analyses of protein and fats degradation, and investigation of sensory and microbial attributes over a three-weeks preservation period. Results indicated that application of 1% garlic oil in combination with 1% chitosan improved preservation and shelf life of Rohu meat, sufficiently safe for human consumption, up to 14 days without exceeding safety limits of all parameters studied. Therefore, it is suggested that the combination of garlic oil and chitosan may be used as an alternate preservative in refrigerated conditions for Rohu fillets preservation and shelf life extension.

Introduction

Fish is a valuable food containing easily digestible high quality proteins, vitamins, minerals, antioxidants and long chain poly unsaturated fatty acids. However, high moisture content and a low pH render it extremely perishable product^1,2. Rohu (Labeo rohita) is a widely distributed fish in south Asia. It belongs to carp family, with many common names locally, including Rohu and Dhambra³. It has strong regional and economic importance as it is naturally distributed in the freshwater bodies of Pakistan, India Bangladesh and Myanmar⁴. It is one of the most commonly consumed and preferably cultured fish in the region due to its taste, nutritional value, affordable price, and high yield^5,6. It is a column feeder and feeds mostly on plankton and weeds in the middle layer. Therefore, it can be easily cultured in polyculture with other economically important carps. Rohu has considerable local market with size range form 2–3 kg and pricing range US$ 1.5–2.0 per kg⁷. Both India and Bangladesh being major producer of the carp, are also increasing culturing of Rohu due to consumer demand, market value and high yield⁸. Safe storage and supply of fish and fish products is crucial to maintain its shelf life, texture, taste and nutritional value to fulfill the demands of tremendously expanding human population. This is an active research area with objectives to improve the preservation methods and techniques⁹.

Meat spoilage mainly occurs due to chemical, enzymatic and microbial activities¹⁰. Among above mentioned modes, microbial spoilage contributes about 30% loss alone^11,12. Enzymatic autolytic spoilage on the other hand is responsible for the damage of fish meat texture^13–15. Chemical degradation includes oxidation of lipid that render unpleasant fish taste¹⁶. Degradation compromises safety and food quality that directly implicates economic loss and rise in food waste¹⁷. Synthetic preservatives though control the rancidity effectively, but in the recent years, safety concern has extensively established market trends to search for natural preservatives as an alternate option¹⁸. Furthermore, fast life today also encourages the consumption of readily available preserved food. Therefore, to ensure the health safety different natural substances having antimicrobial antioxidant properties are being used as preservatives¹⁹.

Natural preservatives have been reported from a number of potential sources e.g. plants, animals, bacteria, algae and fungi^20,21 which have shown remarkable effects on antimicrobial activities, decreasing lipid oxidation and increasing shelf life of fish meat and its other products²². Appropriate use of essential oils and extracts of different plant sources (e.g. Garlic, coriander, onion, rosemary, turmeric, ginger and thyme) as natural preservatives is emerging to control rancidity and microbial contamination in storage conditions^1,23. Garlic (Allium sativum) is a common house hold with well-known medicinal history that is used to enhance the flavor of the fish meat^24,25. Allicin and other hydrophobic compounds of garlic (e.g. vinyldithiins, ajoenes, and diallyl polysulfides) are responsible for its antioxidant, antimicrobial, anti-fungal and antiviral properties^26–29. Shelf life enhancement in different fish species of global importance has been investigated under storage conditions using garlic essential oil alone and in combination with other essential oils^22,30–32. Similarly, chitosan, a natural biopolymer obtained by the deacetylation of chitin has antimicrobial, antioxidant potential and film forming properties that can lock the moisture and acts as gas barrier. Therefore, may be used in combination with essential oils as edible coating for meat of different origins where it serves as a vehicle for these bioactive compounds^33–35. These bio-edible materials are emerging as future preservative materials³⁶.

Post-harvest processing has been considered as key exercise for transformation of fish product into economic value addition. Unfortunately, about 60% of total fish production is wasted as trash and used in fishmeal production in Pakistan which is a low value product. Furthermore, due to outdated processing major part of export is in the form of frozen fish instead of finished product like fillets. The European Union (EU) banned Pakistani fish products for six years (from 2007 onward) due to failure to meet EU sanitary conditions. The recent growth of supermarket and catering business in Pakistan, import of fish fillets has increased from 5.4 to 17.7 million US$³⁷. This shows an incompetency of local products to cover even the local market demand due to low-quality processing. Keeping in view the huge potential of fish products in local super market industry, we designed this study to investigate the effect of garlic oil and chitosan, as natural edible preservatives, on shelf life of Rohu meat in refrigerated conditions. It is being proposed that garlic oil in combination with chitosan can considerably enhance the shelf stability of Rohu (Labeo rohita) meat, rendering it suitable for extended supermarket display and ready-to-use applications. Nevertheless, findings of the present study would ultimately lead to improve the food security and protein shortage in the country.

Materials and methods

Fish samples and experimental design

The Rohu fish (1 ± 0.5 kg average weight) was purchased alive from Head Qadirabad commercial fish market (Latitude, 32° 17’55.7"N, Longitude 73° 41’ 35.6"E) and then was beheaded, gutted and cleaned. The analysis was carried out immediately after harvesting. Four treatment lots (pooled samples) (100 g) were assigned and two-factor factorial CRD experimental design was followed³⁸. Each treatment was carried out with n = 3 independent biological and n = 3 independent technical replicates. The meat samples were treated with chitosan and garlic oil as 0% & 1% in interaction. The vacuum packing of the samples was done in zip-locked polyethylene bags after treatment, labeled and stored at 4 °C. Subsequently, analyses were done at day 0, 7, 14 and 21 of storage.

Proximate composition analysis

The proximate composition was evaluated by the determination of crude fat, moisture, protein and ash by following standard methods of AOAC. Briefly, muscle samples were homogenized well with mortar and pestle. Crude fat and protein were determined by using soxhlet³⁹ and Kjeldahl apparatus⁴⁰, respectively. The moisture contents were determined by oven drying the samples for 12 h at 105 °C.

Water-holding capacity (WHC)

Muscles of Labeo rohita (5 g), minced in mortar and pestle and salt-induced method was used for WHC determination⁴¹. Each sample was centrifuged at 10,000 ×g for 15 min at room temperature and liquid was drained by inverting tubes for 30 min. The percentage weight gain of pellet was expressed as WHC.

Protein extractability

After homogenization of minced muscle (2.5 g) with 25 ml of Tris–HCl (0.03 M) the homogenate was centrifuged at 7,000 ×g for 15 min at 4 °C. After separation of supernatant, remaining residues was again processed as mentioned above. The water extractable proteins (WEP) were determined in total 15 ml supernatant recovered. The remaining residual muscle material was used for salt extractable proteins (SEP) by homogenizing with 25 ml of Tris–HCl (0.03 M) and KCl (0.6 M) at pH 7³⁸ with above mentioned method and protein concentration was determined by using Biuret method⁴².

Total volatile basic nitrogen (TVBN)

To determine TVBN, micro diffusion method was used by homogenizing fish sample (2 g) with Magnesium oxide (MgO) followed by distillation⁴³. After collecting distillate in a flask containing mixed indicator (methylene blue + methyl red) and 3% of boric acid (H₃BO₃), was incubated for color change (purple to green). Collection was continued for 2 times after colour change. Afterward distillate titration against 0.05 M of sulfuric acid was carried out and the consumed volume of sulfuric acid showed TVB-N content (mg/100 g).

Peroxide value (POV)

The POV was determined by spectrophotometry⁴⁴. Ether extraction and distillation was used to extract and purify fat contents from fish samples³⁸. After purification 10 mg fat sample was dissolved in 9.8 ml mixture of chloroform-methanol (7:3, v/v), followed by addition of 0.2 ml ammonium thiocyanate (30% v/v) and 50 µl of FeCl₂ (1 M). After 10 min, absorbance of this mixture was measured at 500 nm through double beam spectrophotometer.

Thiobarbituric acid reactive substances (TBARS)

The measurement of TBARS was done by homogenization of sample in 3 ml Tris-maleate (80 mM, pH 7.4) and KCl (0.15 M)⁴⁵. Briefly, lipid peroxidation was done by adding ascorbic acid to the sample and incubated at 37 °C for 30 min. HCl (0.7 M) and Thiobarbituric acid (TBA) 5 ml each were added followed by mixing with sample and boiled for 25 min. After cooling 5 ml of trichloroacetate (TCA) was further added and tubes were centrifuged at 495 xg for 5 min. Absorbance was measured at 530 nm using spectrophotometer and TBARS contents were estimated in terms of µg malondialdehyde (MDA) equivalents/mg tissue using standard solution of MDA.

Total viable count (TVC)

The TVC was carried out through spread plate method. Serial sample (0.1 ml) dilution was speeded on agar plate and subsequently colony forming units (CFU) were counted after overnight aerobic incubation at 37 °C. The counted CFU/g was converted to Log₁₀ CFU/g^38,46.

Sensory assessment

Highly qualified seven-member panel was requested to evaluate blind coded fish samples by using Quality Index Method (QIM) scheme through 10-point Hedonic scale. The assessment was based on considering firmness (1 extremely soft; 10 very firm), texture (1 very tender; 10 firm), odor (1 off odor/undesirable, 10 highly desirable)⁴⁷. The sensory score below three was considered as rejection of the sample in terms of shelf life criteria.

Statistical analysis

The CoStat computer package (Version 6.303, PMB320 Monterey, CA, 93940 USA) was used to analyze the observed data statistically. Two-way analysis of variance (ANOVA) was applied to determine the impact of treatments and shelf life assessment for meat samples. The p < 0.001 value was considered as significant for all analyses.

Results

Garlic oil and chitosan separately and in combination was applied on Rohu fish meat to enhance its self-life in refrigerated conditions. Standard preservation parameters included moisture contents, crude proteins, crude lipids, water and salt extractable proteins, water holding capacity, TBARS, peroxide value and total volatile basic nitrogen were determined. Similarly, sensory attributes including firmness, texture and odor were also analyzed. As a whole, our treatments showed significant effect on above mentioned parameters showing potential of garlic oil and chitosan as alternative organic preservative candidate for fish meat preservation.

Analysis of the proximate composition

The decrease in moisture contents with the passage of time in control and treatments was found as a general trend. However, this decrease was significantly (p < 0.001) less in garlic oil and chitosan treatments (74.5 ± 0.02, 74.6 ± 0.01) compared to control group (74.5 ± 0.01). The treatment of chitosan alone and in combination with garlic oil demonstrated least change (74.6 ± 0.01, p < 0.001) and was found the most effective treatment showing 0.2% improved moisture retention compared to control (Table 1). The percentage of crude proteins was observed constant for one week of preservation but it was significantly decreased in second and third week in all groups. All the treatments depicted significantly (p < 0.001) less decrease with the passage of time compared to control group (18.4 ± 0.01). Combination of garlic oil and chitosan revealed 0.5% less decrease (18.5 ± 0.01) relative to control and stood most effective among all treatment (Table 1). Overall decrease (p < 0.001) in crude lipid contents was witnessed in each of the treatment from day 0 to day 21. Interestingly, garlic oil (4.7 ± 0.01) and chitosan (4.8 ± 0.01) alone as well as in combination (4.8 ± 0.01) exhibited similar results with improved (0.32%) crude lipid contents relative to control (4.6 ± 0.01) (Table 1).

Table 1.

Effects of garlic oil and chitosan on proximate composition of Rohu meat in refrigerated conditions.

Parameters	Treatments	Day 0	Day 7		Day 14	Day 21
Moisture (%)	Control	74.8 ± 0.01 A	74.6 ± 0.01B		74.5 ± 0.02 C	74.5 ± 0.01Dc
	Garlic oil	74.8 ± 0.01 A	74.7 ± 0.02B		74.6 ± 0.01 C	74.5 ± 0.02Db
	Chitosan	74.8 ± 0.01 A	74.7 ± 0.02B		74.7 ± 0.01B	74.6 ± 0.01Ca
	Garlic oil x Chitosan	74.8 ± 0.01 A	74.7 ± 0.01AB		74.7 ± 0.08AB	74.6 ± 0.01Ba
Crude protein (%)	Control	18.6 ± 0.02 A	18.5 ± 0.01 A		18.5 ± 0.01Bc	18.4 ± 0.01Cc
	Garlic oil	18.5 ± 0.01 A	18.5 ± 0.01 A		18.5 ± 0.01Bb	18.5 ± 0.01Cb
	Chitosan	18.6 ± 0.01 A	18.5 ± 0.01 A		18.5 ± 0.01Bab	18.5 ± 0.01Cb
	Garlic oil x Chitosan	18.6 ± 0.01 A	18.5 ± 0.01 A		18.5 ± 0.00Bc	18.5 ± 0.01Ba
Crude Lipid (%)	Control	4.9 ± 0.01 A	4.8 ± 0.02B		4.7 ± 0.01Cc	4.6 ± 0.01Db
	Garlic oil	4.9 ± 0.01 A	4.8 ± 0.01AB		4.8 ± 0.01Bb	4.7 ± 0.01Ca
	Chitosan	4.9 ± 0.01 A	4.8 ± 0.01B		4.8 ± 0.01Cab	4.8 ± 0.01Da
	Garlic oil x Chitosan	4.9 ± 0.01 A	4.9 ± 0.01 A		4.8 ± 0Ba	4.8 ± 0.01Ca
ANOVA (p-values)
	Treatments	Time		Time x treatment
Moisture	< 0.001	< 0.001		0.004
Crude protein	< 0.001	< 0.001		0.002
Crude lipid	< 0.001	< 0.001		< 0.001

Note: Means of the data were compared using Ducan Multiple Range Test. Different uppercase superscripts in a row indicate a significant effect of storage duration and different lowercase superscripts in a column indicate significant difference of treatments.

Proteins extractability

A linear increase (p < 0.001) in water extractable proteins value was found from day 0 to day 21. Significantly (p < 0.001) low decrease in experimental group was observed with the passage of time relative to control. However, significantly (p < 0.001) least change was observed in combined treatment of garlic oil and chitosan (7.03 ± 0.01) indicating 3.5% improved protein extractability value (Table 2). Similarly, salt extractable proteins depicted decreasing trend from day 0 to 21 in all groups. However, significantly (p < 0.001) less decrease was observed in the experimental groups compared to control (13.8 ± 0.02). Furthermore, among treatments chitosan alone (14.2 ± 0.01) and in combination with garlic oil (14.3 ± 0.01) demonstrated similar results with improved (3%) protein extractability (Table 2).

Table 2.

Effects of garlic oil and chitosan on water, salt extractable proteins and water holding capacity of refrigerated Rohu meat.

Parameters	Treatments	Day 0	Day 7		Day 14	Day 21
Water extractable proteins (g/100 g wet weight)	Control	6.4 ± 0.02D	6.8 ± 0.01Ca		7.04 ± 0.02Ba	7.3 ± 0.01Aa
	Garlic oil	6.5 ± 0.02D	6.8 ± 0.01Cb		7.0 ± 0.01Bb	7.2 ± 0.01Ab
	Chitosan	6.5 ± 0.02D	6.8 ± 0.02Cc		6.9 ± 0.01Bc	7.1 ± 0.01Ac
	Garlic oil x Chitosan	6.5 ± 0.01D	6.6 ± 0.00Cd		6.73 ± 0.01Bd	7.0 ± 0.01Ad
Salt extractable proteins (g/100 g wet weight)	Control	14.5 ± 0.01 A	14.2 ± 0.01Bc		14.05 ± 0.01Cd	13.8 ± 0.02Dc
	Garlic oil	14.5 ± 0.01 A	14.3 ± 0.02Bc		14.1 ± 0.01Cc	14 ± 0.02Db
	Chitosan	14.6 ± 0.02 A	14.4 ± 0.01Ba		14.3 ± 0.01Cb	14.2 ± 0.01Da
	Garlic oil x Chitosan	14.5 ± 0.01 A	14.5 ± 0.02Ba		14.4 ± 0.01Ca	14.3 ± 0.01Da
Water holding capacity (%)	Control	7.7 ± 0.01 A	7.4 ± 0.02Bd		7.4 ± 0.03Bb	7.0 ± 0.01Cd
	Garlic oil	7.8 ± 0.02 A	7.5 ± 0.01Bc		7.4 ± 0.01Cc	7.2 ± 0.03Dc
	Chitosan	7.8 ± 0.01 A	7.6 ± 0.02Bb		7.4 ± 0.02Cb	7.2 ± 0.02Db
	Garlic oil x Chitosan	7.8 ± 0.01 A	7.7 ± 0.01Ba		7.5 ± 0.01Ca	7.4 ± 0.02Da
ANOVA (p-values)
	Treatments	Time		Time x treatment
Water extractable proteins	< 0.001	< 0.001		< 0.001
Salt extractable proteins	< 0.001	< 0.001		< 0.001
Water holding capacity	< 0.001	< 0.001		< 0.001

Note: Means of the data were compared using Ducan Multiple Range Test. Different uppercase superscripts in a row indicate a significant effect of storage duration and different lowercase superscripts in a column indicate significant difference of treatments.

Water holding capacity

A decrease in water holding capacity from day zero to 21st was observed as a general trend in all groups. But treatment groups especially in combination form depicted significantly less change (7.4 ± 0.02) compared to control group (7.0 ± 0.01) and indicated 6.7% better water holding capacity (Table 2).

Thiobarbituric acid reactive substances (TBARS)

Our study revealed an enhancement of antioxidant capacity compared to control (1.43 ± 0.01) in all the treatment groups. At day 7 chitosan alone and garlic oil x chitosan displayed same effect but afterward, synergistic effect of garlic oil and chitosan (1.16 ± 0.01) seemed to maintain a slower decline in lipid deterioration, which is evident in lower TBARS values among all treatment groups generally and 18.9% lower than control group (Table 3).

Table 3.

Effects of garlic oil and chitosan on TBARS, peroxide value and total volatile basic nitrogen Labeo rohita meat preserved in refrigerator.

Parameters	Treatments	Day 0	Day 7		Day 14	Day 21
TBARS (mg MDA-eq/kg meat)	Control	0.83 ± 0.01D	1.08 ± 0.01Ca		1.26 ± 0.03Ba	1.43 ± 0.01Aa
	Garlic oil	0.84 ± 0.01D	0.99 ± 0.01Cb		1.16 ± 0.01Bb	1.34 ± 0.02Ab
	Chitosan	0.84 ± 0.01D	0.94 ± 0.02Cc		1.11 ± 0.01Bc	1.26 ± 0.01Ac
	Garlic oil x Chitosan	0.83 ± 0.01D	0.90 ± 0.01Cc		0.97 ± 0.01Bd	1.16 ± 0.01Ad
Peroxide value (meq of O2/kg fats)	Control	8.67 ± 0.02D	10.2 ± 0.07Ca		11.66 ± 0.04Ba	12.77 ± 0.02Aa
	Garlic oil	8.64 ± 0.03D	9.57 ± 0.02Cb		10.66 ± 0.03Bb	11.26 ± 0.01Ab
	Chitosan	8.65 ± 0.01D	9.26 ± 0.01Cc		10.62 ± 0.01Bb	11.13 ± 0.02Ac
	Garlic oil x Chitosan	8.65 ± 0.03D	9.05 ± 0.01Cd		10.14 ± 0.01Bc	10.84 ± 0.02Ad
Total Volatile Basic Nitrogen (mg/100 g fish meat)	Control	16.36 ± 0.03D	18.26 ± 0.01Ca		19.54 ± 0.03Ba	20.77 ± 0.02Aa
	Garlic oil	16.38 ± 0.01D	17.77 ± 0.02Cb		18.57 ± 0.01Bb	19.77 ± 0.02Ab
	Chitosan	16.36 ± 0.01D	17.57 ± 0.01Cc		18.26 ± 0.01Bc	19.47 ± 0.02Ac
	Garlic oil x Chitosan	16.36 ± 0.01D	17.16 ± 0.01Cd		18.03 ± 0.01Bd	19.26 ± 0.02Ad
ANOVA (p-values)
	Treatments	Time		Time x treatment
TBARS	< 0.001	< 0.001		< 0.001
Peroxidase value	< 0.001	< 0.001		< 0.001
Total Volatile Basic Nitrogen	< 0.001	< 0.001		< 0.001

Note: Means of the data were compared using Ducan Multiple Range Test. Different uppercase superscripts in a row indicate a significant effect of storage duration and different lowercase superscripts in a column indicate significant difference of treatments.

Peroxide value (POV)

Initially all the treatment and control groups showed lipid deterioration evident by rising POV and this value kept rising in control till day 21st. On the other hand, a significant (p < 0.001) prevention in lipid loss was observed in experimental groups (combination of garlic oil and chitosan) with least peroxide values (Table 3). In this context combination of garlic oil and chitosan demonstrated 15% less lipid deterioration compared to control.

Total volatile basic nitrogen (TVBN)

Our study indicated that TVB-N values increased with the course of time in all treated groups. This value remained significantly (p < 0.001) high in control groups (20.77 ± 0.02) till day 21st as compared to experimental groups. Furthermore, the lowest TVB-N values (19.26 ± 0.02) at day 21 were observed in samples treated with combined chitosan and garlic oil showing 7.2% improved preservation (Table 3).

Sensory attributes analysis

Sensory attributes including firmness, texture and odor were analyzed from day 0 to 21st. The sensory acceptability score for all sensory attributes were analyzed and significant decrease was observed with passage of time for all treatment groups. Furthermore, treatment groups significantly (p < 0.001) indicated better preservation values compared to untreated control group, for firmness, texture and odor (5.26 ± 0.03, 5.57 ± 0.01, 6.03 ± 0.02). Among all treatment groups’ combination of garlic oil and chitosan (7.4 ± 0.03, 7.84 ± 0.02, 7.87 ± 0.03) showed highest sensory score for all of the attributes (day 0 to 21) showing promising treatment effect (Table 4). In conclusion, garlic oil and chitosan combination significantly enhanced sensory attributes, resulting in improved firmness 42.3%, texture 40.7% and odor 30.5% compared to the control group.

Table 4.

Effects of garlic oil and chitosan on sensory attributes of refrigerated Rohu meat.

Parameters	Treatments	Day 0	Day 7		Day 14	Day 21
Firmness	Control	9.98 ± 0.04Aa	8.47 ± 0.02Bd		6.27 ± 0.02Cd	5.26 ± 0.03Dd
	Garlic oil	9.72 ± 0.04Ab	8.88 ± 0.02Bc		7.56 ± 0.03Cc	6.6 ± 0.02Dc
	Chitosan	9.98 ± 0.04Aa	9.05 ± 0.02Bb		7.84 ± 0.01Cb	6.79 ± 0.01Db
	Garlic oil x Chitosan	9.98 ± 0.04Aa	9.14 ± 0.01Ba		8.14 ± 0.01Ca	7.4 ± 0.03Da
Texture	Control	9.77 ± 0.02Ab	8.57 ± 0.01Bd		6.87 ± 0.03Cd	5.57 ± 0.01Dd
	Garlic oil	9.98 ± 0.04Aa	8.85 ± 0.01Bc		7.17 ± 0.01Cc	6.53 ± 0.01Dc
	Chitosan	9.97 ± 0.05Aa	8.92 ± 0.01Bb		7.54 ± 0.01Cb	6.83 ± 0.02Db
	Garlic oil x Chitosan	9.82 ± 0.04Ab	9.68 ± 0.01Ba		8.07 ± 0.01Ca	7.84 ± 0.02Da
Odor	Control	9.94 ± 0.09 A	8.37 ± 0.03Bd		7.64 ± 0.02Cd	6.03 ± 0.02Dd
	Garlic oil	9.98 ± 0.04 A	8.68 ± 0.01Bc		7.87 ± 0.02Cc	6.37 ± 0.03Dc
	Chitosan	9.92 ± 0.06 A	8.75 ± 0.02Bb		8.14 ± 0.03Cb	7.53 ± 0.02Db
	Garlic oil x Chitosan	9.95 ± 0.07 A	8.86 ± 0.01Ba		8.64 ± 0.01Ca	7.87 ± 0.03Da
ANOVA (p-values)
	Treatments	Time		Time x treatment
Firmness	< 0.001	< 0.001		< 0.001
Texture	< 0.001	< 0.001		< 0.001
Odor	< 0.001	< 0.001		< 0.001

Note: Means of the data were compared using Ducan Multiple Range Test. Different uppercase superscripts in a row indicate a significant effect of storage duration and different lowercase superscripts in a column indicate significant difference of treatments.

Microbiological analysis

Figure 1 shows the effect of garlic oil and chitosan treatment on TVC of Rohu fish meat preserved in refrigerated conditions. It is observed that number of colony forming units per gram (CFU/g) were increased during the preservation from day 0 to day 21st in all treated groups, but this increase was comparatively low in treatment groups as compared to control group (4–8.4 log CFU/g). The treatment groups showed TVC values in range of 4 to 5.4 log CFU/g from day 0 to 14th while it increased up to 6.4 log CFU/g at day 21st. Furthermore, least TVC profile was found in 1% GO treatment (4.02–6.20 log CFU/g) followed by treatment 1% garlic oil x 1% chitosan combination (4.07–6.25 log CFU/g) (Fig. 1).

Fig. 1.

Effect of garlic oil and chitosan on total viable count (TVC) of preserved Rohu meat. GO = garlic oil, Ch = chitosan.

Discussion

Deterioration of muscle tissue as a result of protein degradation, lipid oxidation and microbial activities, is the main cause of significant economic loss by reducing the shelf life of meat^48,49. Natural organic preservatives are being demanded these days due to toxic outcomes of synthetic preservatives which are reported to pose very serious food security and public health risks^50,51.

Proximate analysis is one of the general qualitative assessments to check the quality of food. The application of chitosan alone and in combination with garlic oil established as most effective natural preservatives with same effect allowing least changes (p < 0.001) in moisture contents during the course of preservation (Table 1). The significant preservative role of chitosan is due to its coating effect, to contain moisture contents. Our findings are in line with da Silva Santos 2017⁵² who reported similar effect of chitosan coating with liquid smoking in prevention of water loss from Nile tilapia (Oreochromis niloticus) fillets. The least decrease in crude proteins percentage value was observed with garlic oil and chitosan (in combination) in all treatment groups and hence proved most effective (lower degradation rate) among all treatments (Table 1). This may be attributed to restriction of autolytic damage and microbial activities by synergistic effect of blend of garlic oil and chitosan⁵³. Interestingly, in case of crude fats garlic oil and chitosan alone as well as in combination displayed analogous effects. The antioxidant properties of garlic oil preserved lipids by lowering the fatty acid oxidation rate³². On the other hand, film forming properties of chitosan act as oxygen barrier that reduce lipid degradation and microbial growth as well^52,54.

Proteins extractability measures the muscle tissue deterioration which further results into loss of water holding capacity of meat. Least change in water extractable protein value was revealed in combined treatment of garlic oil and chitosan (Table 2). These results are in accordance with the findings of Nawaz et al. (2020)³⁸. In this investigation less decrease in salt extractable proteins was found in the treatment groups compared to experimental control. Additionally, chitosan independently and in interaction with garlic oil demonstrated similar result with highest value (p < 0.001) (Table 2). This slow decline in treatment groups shows strong antioxidant properties of garlic oil and coating effect of chitosan which blocks oxygen supply, hence preventing protein damage^55,56. There was least decrease in water holding capacity, among all treated groups (Table 2).

Lipid deterioration is the major limiting factor as it gives off flavors to the stored fish, turning it rancid⁵⁷. MDA is taken as measure of rancidity which is a reaction product of poly unsaturated fatty acids and oxygen⁵⁸. The synergistic effect of garlic oil and chitosan seemed to maintain a slower decline in lipid deterioration, which is evident in lower TBARS values⁵⁹. Flavonoids, Phenolic and sulfur containing compounds of garlic oil and film forming and metal chelating properties of chitosan might have joined effect to work as free radical scavengers. Foromandi and Khani (2023)⁶⁰ have reported an increase in the antioxidant ability of chitosan when used in combination with garlic extracts and coriander essential oil and chitosan alone in refrigerated trout fillets. In addition, Marcu et al. (2022)⁶¹ proposed garlic extracts as an efficient tool to control lipid oxidation by obtaining lower TBARS values compared to control. Similarly, Demircan et al. (2020)⁶² used chitosan coating with lemon extracts on mackerel fillets and declared it as antioxidant preservative. Furthermore, peroxides formation during storage of meat due to oxidation of unsaturated fatty acids, making meat unpleasant, is determined through peroxide value⁶³. Bacterial enzymes which are sensitive to free fatty acid oxidation produce peroxides and raise POV during the preservation process^6,64. Initially all the treatment and control groups presented lipid deterioration evidenced by rising POV, but this value kept rising in control and a significant prevention of lipid loss was observed in treated groups (chitosan and garlic oil combination) having least peroxide values^34,63,65,66. Nawaz et al. (2020)³⁸ also reported similar results with chitosan and rosemary in refrigerated Cirrhinus mrigala samples. Other studies showing low POV in chitosan treated samples compared to control have also been documented^67,68.

Degradation of proteins and other nitrogenous compounds, including nucleotides, cause the production of organic amines which are called as total volatile basic nitrogen (TVB-N)⁶⁹. TVB-N has been adapted as one of the major quality indicators used to assess the freshness of fish under storage conditions⁷⁰. It quantifies the amount of ammonia and other amines produced by deamination of nucleotides and amino acids by the activity of enzymes and bacteria⁷¹. Present study indicated that TVB-N values increased with the course of time which is in accordance with the findings of Nawaz et al. 2020³⁸ and Wang, et al. (2017)⁷². Lower TVB-N values at the end of the experiment were related to treatment groups with lowest values in chitosan and garlic oil interaction whereas highest were associated to non-treated group. The threshold value for acceptability is 25 g N/100 g flesh⁷³, neither of the group reached the highest limit, however the significant decline in TVBN values in chitosan and garlic oil interaction strongly suggests the antimicrobial action of garlic. Garlic due to its components like allicin, inhibits bacterial growth and reduces the rate of protein deterioration. Chitosan on the other hand effectively seals oxygen hence limiting bacterial growth, and hinder the breakdown of proteins^74,75. Wang et al. (2017) ⁷² also culminated similar results where garlic oil and chitosan interaction showed lower TVBN values in Ctenophyaryngodon idella (Grass carp) during storage.

Sensory attributes always play a vital role in establishment of consumer acceptability. Its score was decreased from day 0 to day 21st for all of the treatments. Combination of garlic oil and chitosan showed highest sensory score (for all attributes) showing promising treatment effect (Table 4). Similar observations (consumer’s acceptance) were reported by Hussein et al. (2023)³² by the application of garlic oil and coriander essential oil (CEO) on the meat of O. niloticus. Furthermore, garlic oil alone also indicated promising results in this regard^76,77.

Microbial activity is one of the main problems in storage and supply of a perishable product like fish meat². Moreover, high pH and moisture contents may also facilitate bacterial activity⁷⁸. Current study demonstrated that TVC value (CFU/g) was increased from day 0 to day 21st in all of the groups. However, this increase was low in treated groups when compared to control groups (4–8.4 log CFU/g). Similar results were reported by Smyth (2010)⁷⁹ and Chen (2018)⁷⁷ that garlic oil reduced TVC value and restrict the microbial growth during fish meat preservation. Diallyl disulfide in garlic oil showed growth inhibition in Pseudomonas bacteria⁵³. Similarly, Uçak, (2019)⁸⁰ also documented slower increase in bacterial count with the application of garlic peel extracts as compared to control group. According to ICMSF (1986)⁸¹ the upper acceptable limit of TVC is 6–7 log CFU/g. The current study showed that TVC values in all treated groups were in acceptable range (4–6.44 log CFU/g) from day 0 to day 21. Furthermore, least TVC profile was found in 1% garlic oil treatment (4.02–6.20 log CFU/g) followed by treatment 1% garlic oil x 1% Chitosa combination (4.07–6.25 log CFU/g) (Fig. 1).

Conclusion

The combination of garlic oil (1%) and chitosan (1%), among all treatments, demonstrated improved preservation to increase shelf life of Rohu fish meat storage at 4℃ up to 21 days. This may be potentially attributed to synergistic effect of simultaneous application of garlic oil and chitosan as antioxidant and antimicrobial agent, evident by augmented TBARS (18.9%), POV (15%), TVBN (7.2%) and TVC (6.25 log CFU/g) values. Moreover, the blend of 1% garlic oil and 1% chitosan maintained the acceptable range of sensory attributes with improvement in firmness 42.3%, texture 40.7% and odor 30.5% which suggests it preferable for human consumption as edible natural preservatives. The presented data may be helpful as base line data and model for researchers and meat producers to full fill the meat demand of increasing human population through natural preservatives by reducing the spoilage of meat and meat products. The findings of our investigation would particularly be supportive for growing supermarket industry by improving shelf life of perishable fish meat at refrigerated conditions, hence providing ready to use fish products in this fast era. Furthermore, extensive studies are being recommended to investigate the effect of garlic oil and chitosan as natural preservative candidates for other fish species of global food preference as well as other meat types.

Author contributions

A.S. conducted all experimental work, data compilation and analysis, wrote the manuscript. A.M.K. designed the research work, supervised all research work and data analysis, edited and reviewed the manuscript.

Data availability

The datasets were generated or analyzed during this study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.