# Polyphenol-Rich Extracts of Traditional Culinary Spices and Herbs and Their Antibacterial Activity in Minced Beef

Authors: Saeed Akhtar; Muhammad Waseem; Nazir Ahmad; Tariq Ismail; Zulfiqar Ahmad; Muhammad Faisal Manzoor; Azhari Siddeeg
Journal: Journal of Food Quality
 (2019)

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## Abstract

This study was conducted to elucidate minced beef stabilization properties of hydroalcoholic extracts of commonly used culinary spices from Pakistan against meat oxidative stress and microbial spoilage. Hydroalcoholic extracts of six selected spices, namely, onion, ginger, turmeric, coriander, fennel, and mint, were evaluated to inhibit microbial growth in minced beef under refrigerated storage (4°C) of nine days. Maximum phenolic concentration, i.e., 70.8 mg GAE/100 g, and free radical scavenging activity (75.9%) were anticipated by hydromethanolic extracts of ginger. The results propose that the addition of hydroalcoholic extracts of ginger and coriander @ 6.0% anticipate significantly (p<0.05) higher inhibitory effects against Staphylococcus aureus and Escherichia coli. The results of this research conclude that the utilization of hydroalcoholic extracts may serve as a promising approach to preserve microbiological as well as the oxidative quality of minced beef and products of meat origin.


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## Body

## 1. Introduction

Spices have a long history for culinary application as seasoning ingredients in various cultures, e.g., garlic, onion, cinnamon, anise, clove, and red pepper are preferred seasoning agents of Chinese culture while coriander and black pepper are likely consumed in the East Indian region [1]. Spice extracts and essential oils have been extensively explored for shelf stability of raw and processed chicken [2, 3], shallow and deep-fried meat [4], fermented meat [5], meat sausages [6], and dried cured meat [7].Microbiological food safety in the meat distribution system can be achieved to a greater extent with natural ingredients of plants and animal origins like organic acids, plant extracts, and essential oils [8]. Antimicrobial features of spices are predominantly associated with phytochemicals like flavonoids, flavones, isoflavones, and anthocyanins that anticipate significant free radicals and free metal ion binding properties in food systems [9, 10]. Active ingredients of spices have also been attributed to bringing about changes in cellular membrane permeability leading to intracellular matrix leakage and cell lysis [11]. Typical characteristics of spices defining their role as potential antimicrobial agents have been embedded in rendering bacteria to poorly synthesize microbial nucleotides, i.e., DNA and RNA, which could further halt microbial growth and proliferation [12].Spices primarily provide a convenient and reasonable choice towards minimizing household and industrial use of synthetic additives and to add value to the consumer good. Spices could further substantially contribute to reducing the adverse effects of synthetic additives on product quality and consumer’s health. The objectives of the present study entail investigation into assessing the antioxidant potential of turmeric, onion, ginger, fennel, coriander, and mint extracts and their role as antimicrobials in inhibiting microbial growth in the minced beef model under refrigerated storage.


## 2. Material and Methods

### 2.1. Procurement of Raw Materials and Chemicals

Fresh ginger (Zingiber officinale) rhizomes, onion (Allium cepa) bulbs, turmeric (Curcuma longa) stems, coriander (Coriandrum sativum) seeds, fennel (Foeniculum vulgare) seeds, and peppermint (Mentha piperita) leaves were purchased from the local market of Multan, Pakistan. Samples were maintained at −18°C until drying. All reagents unless specified including solvents, sodium acetate buffer (pH = 3.6), DPPH (2, 2-diphenyl-1-picryl-hydroxyl) reagent, Folin–Ciocalteu phenol reagent (FCR), gallic acid, sodium carbonate, TPTZ [2, 4, 6-tri (2-pyridyl)-s-triazine], and ascorbic acid were analytical grade and purchased from Sigma-Aldrich Inc., USA.


### 2.2. Development of Spice Powders

Green spices were procured from the local vegetable market, washed with potable water, sorted, graded, and dehydrated in a cabinet dryer at 70°C to 15–17% moisture contents. Dehydrated spices were ground to 70 mm mesh size, sieved, and stored in airtight plastic containers at 25°C for further applications [13].


### 2.3. Physicochemical Analysis of Spice Powders

Fine powders of spices were analyzed for moisture, fat, ash, and protein contents in accordance with the procedure laid down in AOAC [14, 15]. Carbohydrate contents were estimated as nitrogen-free extract (NFE) using formula, i.e., NFE% = 100 − (moisture + crude protein + total ash + crude fat + crude fiber). Mineral contents (Na, Ca, and K) of spice powders were analyzed using a flame photometer in accordance with the method laid down by AOAC [14].


### 2.4. Total Phenolic Contents

Spice powders were soaked in hydromethanolic and ethanolic solvents (70 : 30) (solvent: distilled water) for 8 hours. Orbital shaking was performed at 40°C for 3 hours. The supernatant was filtered via filter paper no. 41 followed by rotary evaporation at 40°C. Extracts’ concentrates were freeze-dried and stored at −18°C for further assay. Total phenolic contents in spice extracts were determined by the method adopted by Manzoor et al. [16]. Plant extracts (i.e., 100 ppm) were prepared with solvents, i.e., methanol and ethanol. An aliquot of 0.5 mL was transferred to the test tubes followed by the addition of 10-fold diluted FCR. 2 mL of sodium carbonate (7.5%) was added, and samples were subjected to react for 30 min at 25°C. Absorbance was measured spectrophotometrically (UV-Vis 3000, ORI, Germany) at 760 nm using gallic acid standard (10–100 ppm), and results were expressed as mg GAE/100 g.


### 2.5. 2, 2-Diphenyl-1-Picryl-Hydrazyl (DPPH) Assay

Free radical scavenging activity of hydroalcoholic extracts was determined using DPPH assay [17]. Different concentrations of spice extracts ranging from 50–100 ppm were prepared. Aliquots (50–100 μL) were pipetted in labeled test tubes, and the final volume was adjusted to 100 μL with methanol. 5 mL of DPPH reagent (0.1 mM) was added to each test tube. The contents of test tubes were vortexed and incubated for 20 min at 27°C. Absorbance was measured spectrophotometrically at 517 nm. Free radical scavenging activity was calculated using the following formula:(1)radical scavenging activity%=control Abs.−sample Abs.control Abs.×100.


### 2.6. Microbiological Analysis

#### 2.6.1. Bacterial Cultures and Inocula Preparation

Bacterial isolates from minced beef were spread onto specific microbial culture media including mannitol salt agar (Staphylococcus aureus), MacConkey agar (Escherichia coli), and SS agar (Salmonella spp.). Confirmed colonies of each test microorganisms were shifted to phosphate buffer saline and incubated at 37°C for 3–6 hours to achieve 0.5 McFarland turbidity standard.


#### 2.6.2. Antimicrobial Assay (Disc Diffusion Method)

Antimicrobial screening of spice extracts was performed in accordance with the method developed by Adetunde et al. [18]. Microbial cultures vis. S. aureus, E. coli, and Salmonella spp. were evenly spread on Muller Hinton Agar (MHA) plates. Sterilized discs were aseptically placed over the inoculated MHA media plates. Spice extracts (50 μL) of 150 ppm strength were loaded onto the discs. Solvent and standard drugs, i.e., gentamycin and penicillium (20–30 μg), were taken as negative and positive controls, respectively. MHA plates were subjected to incubation at 37°C for 24 hours, and zones of inhibitions (mm) were computed.


#### 2.6.3. Microbiological Inhibition Properties of Spice Extracts in Minced Beef

Freshly purchased minced beef was decontaminated using sodium hypochlorite (20 ppm). Hundred-gram minced beef sample with no decontamination treatment was designated as a negative control. Twenty-five grams of minced beef was marinated with hydromethanolic extracts of spices including onion, turmeric, ginger, coriander, fennel, and mint at the rate of 1.5%, 3%, and 6%. Marinated samples were stomached for 2 min (Stomacher® 400 Circulator). Stomached samples were further inoculated with 100μL (1.5 × 108 CUF/mL∼0.5 McFarland turbidity standard) cultures of S. aureus and E. coli. Microbial spiked minced beef samples were stored at 4 ± 2°C, and total counts of S. aureus and E. coli of minced beef samples were enumerated on 0, 3rd, 6th, and 9th day of storage. Results were expressed as log10 CFU/g [19, 20].


### 2.7. Statistical Analysis

All experiments were performed twice, and the results were expressed as mean ± SD. Data were statistically analyzed with Statistics 8.1 software using a two-way analysis of variance (ANOVA) technique atp<0.05. Means were compared using the least significant difference (LSD) test.


## 2.1. Procurement of Raw Materials and Chemicals

Fresh ginger (Zingiber officinale) rhizomes, onion (Allium cepa) bulbs, turmeric (Curcuma longa) stems, coriander (Coriandrum sativum) seeds, fennel (Foeniculum vulgare) seeds, and peppermint (Mentha piperita) leaves were purchased from the local market of Multan, Pakistan. Samples were maintained at −18°C until drying. All reagents unless specified including solvents, sodium acetate buffer (pH = 3.6), DPPH (2, 2-diphenyl-1-picryl-hydroxyl) reagent, Folin–Ciocalteu phenol reagent (FCR), gallic acid, sodium carbonate, TPTZ [2, 4, 6-tri (2-pyridyl)-s-triazine], and ascorbic acid were analytical grade and purchased from Sigma-Aldrich Inc., USA.


## 2.2. Development of Spice Powders

Green spices were procured from the local vegetable market, washed with potable water, sorted, graded, and dehydrated in a cabinet dryer at 70°C to 15–17% moisture contents. Dehydrated spices were ground to 70 mm mesh size, sieved, and stored in airtight plastic containers at 25°C for further applications [13].


## 2.3. Physicochemical Analysis of Spice Powders

Fine powders of spices were analyzed for moisture, fat, ash, and protein contents in accordance with the procedure laid down in AOAC [14, 15]. Carbohydrate contents were estimated as nitrogen-free extract (NFE) using formula, i.e., NFE% = 100 − (moisture + crude protein + total ash + crude fat + crude fiber). Mineral contents (Na, Ca, and K) of spice powders were analyzed using a flame photometer in accordance with the method laid down by AOAC [14].


## 2.4. Total Phenolic Contents

Spice powders were soaked in hydromethanolic and ethanolic solvents (70 : 30) (solvent: distilled water) for 8 hours. Orbital shaking was performed at 40°C for 3 hours. The supernatant was filtered via filter paper no. 41 followed by rotary evaporation at 40°C. Extracts’ concentrates were freeze-dried and stored at −18°C for further assay. Total phenolic contents in spice extracts were determined by the method adopted by Manzoor et al. [16]. Plant extracts (i.e., 100 ppm) were prepared with solvents, i.e., methanol and ethanol. An aliquot of 0.5 mL was transferred to the test tubes followed by the addition of 10-fold diluted FCR. 2 mL of sodium carbonate (7.5%) was added, and samples were subjected to react for 30 min at 25°C. Absorbance was measured spectrophotometrically (UV-Vis 3000, ORI, Germany) at 760 nm using gallic acid standard (10–100 ppm), and results were expressed as mg GAE/100 g.


## 2.5. 2, 2-Diphenyl-1-Picryl-Hydrazyl (DPPH) Assay

Free radical scavenging activity of hydroalcoholic extracts was determined using DPPH assay [17]. Different concentrations of spice extracts ranging from 50–100 ppm were prepared. Aliquots (50–100 μL) were pipetted in labeled test tubes, and the final volume was adjusted to 100 μL with methanol. 5 mL of DPPH reagent (0.1 mM) was added to each test tube. The contents of test tubes were vortexed and incubated for 20 min at 27°C. Absorbance was measured spectrophotometrically at 517 nm. Free radical scavenging activity was calculated using the following formula:(1)radical scavenging activity%=control Abs.−sample Abs.control Abs.×100.


## 2.6. Microbiological Analysis

### 2.6.1. Bacterial Cultures and Inocula Preparation

Bacterial isolates from minced beef were spread onto specific microbial culture media including mannitol salt agar (Staphylococcus aureus), MacConkey agar (Escherichia coli), and SS agar (Salmonella spp.). Confirmed colonies of each test microorganisms were shifted to phosphate buffer saline and incubated at 37°C for 3–6 hours to achieve 0.5 McFarland turbidity standard.


### 2.6.2. Antimicrobial Assay (Disc Diffusion Method)

Antimicrobial screening of spice extracts was performed in accordance with the method developed by Adetunde et al. [18]. Microbial cultures vis. S. aureus, E. coli, and Salmonella spp. were evenly spread on Muller Hinton Agar (MHA) plates. Sterilized discs were aseptically placed over the inoculated MHA media plates. Spice extracts (50 μL) of 150 ppm strength were loaded onto the discs. Solvent and standard drugs, i.e., gentamycin and penicillium (20–30 μg), were taken as negative and positive controls, respectively. MHA plates were subjected to incubation at 37°C for 24 hours, and zones of inhibitions (mm) were computed.


### 2.6.3. Microbiological Inhibition Properties of Spice Extracts in Minced Beef

Freshly purchased minced beef was decontaminated using sodium hypochlorite (20 ppm). Hundred-gram minced beef sample with no decontamination treatment was designated as a negative control. Twenty-five grams of minced beef was marinated with hydromethanolic extracts of spices including onion, turmeric, ginger, coriander, fennel, and mint at the rate of 1.5%, 3%, and 6%. Marinated samples were stomached for 2 min (Stomacher® 400 Circulator). Stomached samples were further inoculated with 100μL (1.5 × 108 CUF/mL∼0.5 McFarland turbidity standard) cultures of S. aureus and E. coli. Microbial spiked minced beef samples were stored at 4 ± 2°C, and total counts of S. aureus and E. coli of minced beef samples were enumerated on 0, 3rd, 6th, and 9th day of storage. Results were expressed as log10 CFU/g [19, 20].


## 2.6.1. Bacterial Cultures and Inocula Preparation

Bacterial isolates from minced beef were spread onto specific microbial culture media including mannitol salt agar (Staphylococcus aureus), MacConkey agar (Escherichia coli), and SS agar (Salmonella spp.). Confirmed colonies of each test microorganisms were shifted to phosphate buffer saline and incubated at 37°C for 3–6 hours to achieve 0.5 McFarland turbidity standard.


## 2.6.2. Antimicrobial Assay (Disc Diffusion Method)

Antimicrobial screening of spice extracts was performed in accordance with the method developed by Adetunde et al. [18]. Microbial cultures vis. S. aureus, E. coli, and Salmonella spp. were evenly spread on Muller Hinton Agar (MHA) plates. Sterilized discs were aseptically placed over the inoculated MHA media plates. Spice extracts (50 μL) of 150 ppm strength were loaded onto the discs. Solvent and standard drugs, i.e., gentamycin and penicillium (20–30 μg), were taken as negative and positive controls, respectively. MHA plates were subjected to incubation at 37°C for 24 hours, and zones of inhibitions (mm) were computed.


## 2.6.3. Microbiological Inhibition Properties of Spice Extracts in Minced Beef

Freshly purchased minced beef was decontaminated using sodium hypochlorite (20 ppm). Hundred-gram minced beef sample with no decontamination treatment was designated as a negative control. Twenty-five grams of minced beef was marinated with hydromethanolic extracts of spices including onion, turmeric, ginger, coriander, fennel, and mint at the rate of 1.5%, 3%, and 6%. Marinated samples were stomached for 2 min (Stomacher® 400 Circulator). Stomached samples were further inoculated with 100μL (1.5 × 108 CUF/mL∼0.5 McFarland turbidity standard) cultures of S. aureus and E. coli. Microbial spiked minced beef samples were stored at 4 ± 2°C, and total counts of S. aureus and E. coli of minced beef samples were enumerated on 0, 3rd, 6th, and 9th day of storage. Results were expressed as log10 CFU/g [19, 20].


## 2.7. Statistical Analysis

All experiments were performed twice, and the results were expressed as mean ± SD. Data were statistically analyzed with Statistics 8.1 software using a two-way analysis of variance (ANOVA) technique atp<0.05. Means were compared using the least significant difference (LSD) test.


## 3. Results and Discussion

### 3.1. Physicochemical Properties of Spices and Their Extracts

Data on the nutritional composition of spices powder are presented in Tables1 and 2. A significant difference in ash contents was detected in turmeric (6.5%) and mint powder (1.9%). Maximum fat contents were recorded in fennel while coriander depicted the highest concentration of protein. A significantly higher amount of carbohydrates was recorded in onion powder.Table 1
Proximate composition of spice powders on dry weight basis (g/100 g).
Spices
Moisture
Ash
Fat
Protein
Fiber
Carbohydrates†
Onion
9.5 ± 1.3b
3.4 ± 0.5c
1.5 ± 0.7c
2.5 ± 0.6c
2.2 ± 1.59e
80.9 ± 3.3a
Ginger
10.0 ± 0.3b
5.4 ± 0.4b
1.9 ± 0.2c
6.2 ± 0.6b
6.0 ± 2.7cd
70.5 ± 2.7b
Turmeric
8.8 ± 0.7b
6.5 ± 0.6a
3.8 ± 0.2b
4.0 ± 0.7c
4.8 ± 1.0de
72.2 ± 4.0b
Coriander
9.2 ± 0.6b
2.5 ± 0.1cd
6.8 ± 0.3a
13.2 ± 0.3a
14.3 ± 0.9b
63.1 ± 2.0c
Fennel
9.4 ± 0.8b
5.9 ± 0.4b
7.1 ± 0.6a
3.9 ± 0.2c
20.3 ± 0.9a
53.4 ± 0.9c
Mint
12.3 ± 1.0a
1.9 ± 0.9d
1.3 ± 0.2c
2.2 ± 0.2c
8.4 ± 1.6bc
74.0 ± 3.3b
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05. †Calculations on dry weight basis as 100−(Ash + Protein + Fiber + Fat).Table 2
Mineral composition of spice powders on dry weight basis (mg/kg−1).
Spices
Calcium
Potassium
Sodium
Onion
5.0 ± 0.0d
135.0 ± 0.0c
14.3 ± 0.6c
Ginger
6.0 ± 0.0d
229.7 ± 0.6a
16.0 ± 0.0c
Turmeric
6.3 ± 0.6d
169.0 ± 0.0b
16.3 ± 0.6c
Coriander
18.3 ± 2.5c
94.7 ± 4.9e
95.3 ± 5.5a
Fennel
32.0 ± 2.6a
116.3 ± 5.0d
42.7 ± 2.5b
Mint
24.7 ± 1.5b
89.7 ± 0.6e
18.3 ± 0.6c
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05.The appreciable concentration of sodium, calcium, and potassium was observed in coriander, fennel, and ginger powder, respectively (Table2). Average spice consumption from various modes in the Indian subcontinent has been reported around 10 g that can anticipate ∼1.2–8% of daily energy requirements [21]. In addition to create appeal and anticipate functional properties, compositional analysis of spices thus suggests their supplementary role in improving the nutritional value of the finished goods.


### 3.2. Total Phenolic Contents and Antioxidant Activity

The extracts’ yield, total phenolic contents, and antioxidant potential of spices are presented in Table3. Significant (p<0.05) effect of solvent and type of spices was revealed on phenolic recovery. The highest total phenolic contents with a mean value of 70.8 mg GAE/100 g were recovered from ginger followed by turmeric extracts, i.e., 70 mg GAE/100 g, while onion and fennel hydroethanolic extracts were bearing lower phenolics recovery rate, i.e., 36 mg GAE/100 g and 35 mg GAE/100 g, respectively. Relatively lower total phenolic contents were reported in spices by Kumari and Gupta [22] wherein the phenolic recovery rate was in a range between 20–78 mg GAE/100 g. Hydroalcoholic extraction of plant phenolics has variable recovery rates that depend on the type of solvent, combinations of solvents like water : alcohol ratio (70 : 30) and solvent/solid ratio, part of the plant, i.e., leaves, roots, seeds, fruit, flower, and bark, particle size or the surface area of the plant matter, and extraction conditions like pressure (30–250 bar), extraction time (3-4 hours), and extraction temperature, i.e., ∼25°C [23].Table 3
Extracts’ yield, total phenolic contents, and antioxidant potential of spices.
Spices
Solvent
Extracts yield (%)
TPC (mg GAE/100 g)
DPPH (%)
Onion
MeOH
9.5 ± 1.5a
55.9 ± 3.2bc
54.4 ± 3.8e
EtOH
5.9 ± 0.5bc
36.0 ± 4.6hi
43.1 ± 4.4g
Ginger
MeOH
5.5 ± 1.6bc
70.8 ± 3.3a
75.9 ± 3.9a
EtOH
3.2 ± 0.9ef
54.1 ± 3.4bcd
66.3 ± 5.0bc
Turmeric
MeOH
4.8 ± 0.1cd
51.6 ± 1.6cde
61.4 ± 1.9cd
EtOH
6.2 ± 1.2b
42.7 ± 6.0fgh
46.1 ± 4.0fg
Coriander
MeOH
2.4 ± 0.3f
69.8 ± 1.6a
70.5 ± 2.5ab
EtOH
3.1 ± 0.0ef
49.0 ± 0.5def
51.7 ± 1.6ef
Fennel
MeOH
5.4 ± 0.1bc
46.3 ± 5.2efg
56.2 ± 5.2de
EtOH
4.0 ± 0.0de
34.8 ± 4.7i
41.2 ± 1.2g
Mint
MeOH
3.9 ± 0.1de
59.1 ± 5.7b
62.1 ± 3.2c
EtOH
3.0 ± 0.0ef
40.6 ± 2.9ghi
42.6 ± 1.2g
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05. MeOH = hydromethanolic extracts; EtOH = hydroethanolic extracts; TPC = total phenolic content; DPPH = diphenyl picrylhydrazyl.Significantly higher antioxidant properties were observed in the ginger extract in comparison with extracts of other spices under investigation (Table3). Hydroalcoholic extracts yielded higher free radical scavenging properties with ginger followed by turmeric, i.e., 75.9%, while hydroethanolic extracts presented higher DPPH radical scavenging activities for ginger (66.3%) and coriander (51.7%). Higher DPPH free radical scavenging properties of ginger and turmeric correlates with their higher phenolic contents as compared to the onion extract. DPPH free radical scavenging property of ginger extracts has been previously cited between 67–78% [22]. The considerably higher concentration of hydroxyl rich total phenolics and synergistic role of spices extracts could be achieved by their application as additives in meat and meat-based products.


### 3.3. Antimicrobial Screening of Spice and Herb Extracts

The antimicrobial activity of spice extracts against various pathogenic microbes at 150 ppm concentration is presented in Table4. Inhibition zones of various extracts against Gram-negative and Gram-positive bacteria including E. coli, Salmonella spp., and S. aureus were determined for methanolic and ethanolic extracts of ginger, turmeric, onion, coriander, fennel, and mint extracts at 150 ppm concentration (30–40 μg extracts disc). In comparison with gentamycin and penicillin discs, both methanolic and ethanolic extracts of onion generated larger zones of inhibition against the tested pathogens. Onion extracts generated wider inhibition zones, i.e., 17.1 mm, 16.5 mm, and 15.5 mm, for E. coli, S. aureus, and Salmonella spp., respectively. Comparatively lower antimicrobial activities against tested pathogens were reflected by hydroethanolic extracts of mint, fennel, and coriander extracts.Table 4
Antimicrobial activity of spice extracts against various pathogenic microbes at 150 ppm concentration.
Spices
Extracts
Escherichia coli
Staphylococcus aureus
Salmonella spp.
Gentamycin
—
21.5 ± 0.7a
22.0 ± 0.7a
24.0 ± 0.0a
Penicillin
—
19.5 ± 0.7b
20.5 ± 1.4a
23.5 ± 0.7a
Onion
MeOH
17.1 ± 0.6c
16.3 ± 1.1bc
15.5 ± 3.5b
EtOH
14.5 ± 0.7def
16.5 ± 0.7bc
15.3 ± 0.4b
Ginger
MeOH
11.5 ± 0.7hi
14.5 ± 0.7bcde
9.8 ± 0.4f
EtOH
13.5 ± 2.1efg
16.0 ± 1.4bc
11.0 ± 1.4ef
Turmeric
MeOH
14.8 ± 0.4def
15.5 ± 2.1bc
14.0 ± 1.4bcd
EtOH
16.0 ± 1.4cd
15.0 ± 1.4bcd
15.5 ± 0.7b
Coriander
MeOH
15.2 ± 0.2de
15.5 ± 0.7bc
14.3 ± 0.4bcd
EtOH
13.7 ± 0.2efg
14.0 ± 0.0cde
12.0 ± 0.0def
Fennel
MeOH
13.2 ± 0.3fgh
15.0 ± 0.0bcd
14.8 ± 0.4bc
EtOH
12.3 ± 0.4ghi
12.0 ± 1.4e
13.1 ± 0.2bcde
Mint
MeOH
16.0 ± 0.7cd
16.6 ± 1.3b
14.1 ± 0.4bcd
EtOH
11.0 ± 0.7i
12.7 ± 1.4de
12.7 ± 0.2cde
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05. MeOH = hydromethanolic extracts; EtOH = hydroethanolic extracts.


### 3.4. Effect of Extracts’ Supplementation onE. coli Counts in Minced Beef

E. coli counts under refrigeration of minced beef treated with hydromethanolic extracts of spices were estimated in log10 CFU/g during 0–9 days of storage. Interpretation of the data presented in Figure 1 suggests significant (p<0.05) reductions in E. coli counts of minced beef on treatment with varying levels of spice extracts at different storage intervals. In comparison to the negative control where E. coli counts were found to increase from 5.72 log10 CFU/g to 6.29 log10 CFU/g, coriander extracts’ supplementation in E. coli inoculated minced beef presented peak inhibitory properties, i.e., from 4.8 log10 CFU/g to 5 log10 CFU/g, during 9 d refrigerated storage. Around 0.23 log10 CFU/g increase in E. coli counts was observed in minced beef supplemented with 6% ginger extracts as compared to 0.56 log10 CFU/g and 0.60 log10 CFU/g for negative and positive control under similar study conditions. Fennel and mint extracts were also found equally efficacious in inhibiting the pathogenic load of E. coli. Furthermore, methanolic extracts of fennel, mint, and coriander increased the lag period in relation to the normal control. Comparable role of turmeric and onion extracts were noticed against E. coli inhibition. Pearson correlation (r=0.96) shows that the extracts’ amount and storage duration suggested higher E. coli inhibitory properties of spice extracts at an extended amount of supplementation.Figure 1
E. coli inhibitory activity of spice extracts in marinated minced beef under refrigerated (4°C) 9 d storage. NC: negative control, PC: positive control, OM: onion methanolic extracts, GM: ginger methanolic extracts, TM: turmeric methanolic extracts, CM: coriander methanolic extracts, FM: fennel methanolic extracts, and MM: mint methanolic extracts.
(a)
(b)
(c)
(d)
(e)
(f)Antimicrobial activities of spices have been attributed to flavonoids, saponins, glucosinolates, thiosulfinates, and saponins [24]. Ginger bioactive compounds that exhibit antimicrobial activity include ar-curcumin, caryophyllene, ß-sesquiphellandrene, α-farnesene, and zingiberene [25]. Coriander methanolic extracts have been already reported effective against human pathogens including E. coli and Salmonella typhi [26]. An earlier study carried out by Bali et al. [27] endorsed coriander application at the rate of 2–5% in beef sausages to attribute improved meat quality parameters under refrigerated storage for a period of 14 days. The study further suggested coriander application to anticipate ∼1.18 log inhibition of total bacterial count in comparison with normal control during extended refrigerated storage of 21 days. Relatively lesser antibacterial activity of ginger, turmeric, and onion extracts was observed in ground beef that may be associated with poor distribution of spice extracts in a beef matrix. A study conducted by Gupta and Ravishankar [28] revealed that antimicrobial activity of pure pastes of ginger, garlic, and turmeric against E. coli O157:H7 was found higher than that observed in beef, thus suggesting a partial reduction in bactericidal properties of extracts in food system. Ginger extracts have been also reported as proteolytic because they enhance the antimicrobial characteristics against Gram-negative and positive pathogens including E. coli and L. monocytogenes [29].


### 3.5. Effect of Extracts’ Supplementation onS. aureus Counts in Minced Beef

In comparison with both the positive and negative controls, methanolic extracts of tested spices significantly (p<0.05) inhibited S. aureus growth in minced beef during 0–9 days of the study period. Data presented in Figure 2 showed that minced beef marinated with spice extracts at the rate of 1.5–6.0% offered better shelf stability and reduced pathogen growth. Least S. aureus count increment, i.e., 0.24 log10 CFU/g, was observed on 9th day of storage in minced beef marinated with 6% ginger extracts, whereas positive control inoculated with S. aureus at same inoculation levels as of treatment groups was observed with 0.77 log10 CFU/g increase in pathogen counts at the end of the study. Methanolic extracts of coriander and turmeric also delivered pronounced inhibition in S. aureus proliferation during 9 d storage with up to 0.32 and 0.43 log10 CFU/g upsurge for coriander and turmeric extracts, respectively. Fennel, mint, and onion extracts also exhibited significant (p<0.05) S. aureus inhibition in comparison with the normal and positive control (Figure 2).Figure 2
S. aureus inhibitory activity of spice extracts in marinated minced beef under refrigerated (4°C) 9 d storage. NC: negative control, PC: positive control, OM: onion methanolic extracts, GM: ginger methanolic extracts, TM: turmeric methanolic extracts, CM: coriander methanolic extracts, FM: fennel methanolic extracts, and MM: mint methanolic extracts.
(a)
(b)
(c)
(d)
(e)
(f)Ginger extracts have been reported efficacious against both the Gram-positive and Gram-negative bacteria includingS. aureus and E. coli [30]. In an earlier study wherein ginger extracts were applied as natural preservatives in frozen beef sausages, the extracts’ application at the rate of 1.0% was found to increase product shelf stability by significantly inhibiting microbial growth and lipid oxidation [31]. The study in question declares the application of a relatively higher amount of ginger extracts, i.e., 6.0%, to deliver strong microbial inhibitory properties under refrigerated storage. Ginger extracts have also been reported to disrupt and extensively break muscle fibers [32]. This feature enables spice extracts to deliver higher microbicidal activities alongside meat tenderizing properties.The complex composition of minced beef, i.e., carrying a higher amount of lipids, protein, water, and salts, makes it more resistant towards both the synthetic and natural antimicrobials. Hence, higher concentration and amount of spice extracts are desired to offer microbiological inhibitory properties in food in comparison with the microbial growth medium [10]. A study on 43 different spices used in meat-based cuisines of 36 countries concludes that the spices application as food cleanser and their utilization increases in cookeries [33]. Earlier work on spice application in meat-based products suggests palatability of cooked recipes at relatively higher doses of spices than recommended in recent study. Supplementing 10% extracts of myrtle, rosemary, lemon balm, nettle leaves, green tea, and ginger in beef patties, cooked pork meat, and stewed pork had been suggested as organoleptically acceptable [34, 35]. Variability of different spice extracts in presenting microbicidal properties against both E. coli and S. aureus as observed in this study is associated with their varied antimicrobial property-bearing phytochemical profile and growth conditions. Findings from this recent study also suggest relatively higher amount of extract (i.e., 6.0%) supplementation as marinades in meat-based system to offer antimicrobial and other quality enhancement and preservative properties.


## 3.1. Physicochemical Properties of Spices and Their Extracts

Data on the nutritional composition of spices powder are presented in Tables1 and 2. A significant difference in ash contents was detected in turmeric (6.5%) and mint powder (1.9%). Maximum fat contents were recorded in fennel while coriander depicted the highest concentration of protein. A significantly higher amount of carbohydrates was recorded in onion powder.Table 1
Proximate composition of spice powders on dry weight basis (g/100 g).
Spices
Moisture
Ash
Fat
Protein
Fiber
Carbohydrates†
Onion
9.5 ± 1.3b
3.4 ± 0.5c
1.5 ± 0.7c
2.5 ± 0.6c
2.2 ± 1.59e
80.9 ± 3.3a
Ginger
10.0 ± 0.3b
5.4 ± 0.4b
1.9 ± 0.2c
6.2 ± 0.6b
6.0 ± 2.7cd
70.5 ± 2.7b
Turmeric
8.8 ± 0.7b
6.5 ± 0.6a
3.8 ± 0.2b
4.0 ± 0.7c
4.8 ± 1.0de
72.2 ± 4.0b
Coriander
9.2 ± 0.6b
2.5 ± 0.1cd
6.8 ± 0.3a
13.2 ± 0.3a
14.3 ± 0.9b
63.1 ± 2.0c
Fennel
9.4 ± 0.8b
5.9 ± 0.4b
7.1 ± 0.6a
3.9 ± 0.2c
20.3 ± 0.9a
53.4 ± 0.9c
Mint
12.3 ± 1.0a
1.9 ± 0.9d
1.3 ± 0.2c
2.2 ± 0.2c
8.4 ± 1.6bc
74.0 ± 3.3b
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05. †Calculations on dry weight basis as 100−(Ash + Protein + Fiber + Fat).Table 2
Mineral composition of spice powders on dry weight basis (mg/kg−1).
Spices
Calcium
Potassium
Sodium
Onion
5.0 ± 0.0d
135.0 ± 0.0c
14.3 ± 0.6c
Ginger
6.0 ± 0.0d
229.7 ± 0.6a
16.0 ± 0.0c
Turmeric
6.3 ± 0.6d
169.0 ± 0.0b
16.3 ± 0.6c
Coriander
18.3 ± 2.5c
94.7 ± 4.9e
95.3 ± 5.5a
Fennel
32.0 ± 2.6a
116.3 ± 5.0d
42.7 ± 2.5b
Mint
24.7 ± 1.5b
89.7 ± 0.6e
18.3 ± 0.6c
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05.The appreciable concentration of sodium, calcium, and potassium was observed in coriander, fennel, and ginger powder, respectively (Table2). Average spice consumption from various modes in the Indian subcontinent has been reported around 10 g that can anticipate ∼1.2–8% of daily energy requirements [21]. In addition to create appeal and anticipate functional properties, compositional analysis of spices thus suggests their supplementary role in improving the nutritional value of the finished goods.


## 3.2. Total Phenolic Contents and Antioxidant Activity

The extracts’ yield, total phenolic contents, and antioxidant potential of spices are presented in Table3. Significant (p<0.05) effect of solvent and type of spices was revealed on phenolic recovery. The highest total phenolic contents with a mean value of 70.8 mg GAE/100 g were recovered from ginger followed by turmeric extracts, i.e., 70 mg GAE/100 g, while onion and fennel hydroethanolic extracts were bearing lower phenolics recovery rate, i.e., 36 mg GAE/100 g and 35 mg GAE/100 g, respectively. Relatively lower total phenolic contents were reported in spices by Kumari and Gupta [22] wherein the phenolic recovery rate was in a range between 20–78 mg GAE/100 g. Hydroalcoholic extraction of plant phenolics has variable recovery rates that depend on the type of solvent, combinations of solvents like water : alcohol ratio (70 : 30) and solvent/solid ratio, part of the plant, i.e., leaves, roots, seeds, fruit, flower, and bark, particle size or the surface area of the plant matter, and extraction conditions like pressure (30–250 bar), extraction time (3-4 hours), and extraction temperature, i.e., ∼25°C [23].Table 3
Extracts’ yield, total phenolic contents, and antioxidant potential of spices.
Spices
Solvent
Extracts yield (%)
TPC (mg GAE/100 g)
DPPH (%)
Onion
MeOH
9.5 ± 1.5a
55.9 ± 3.2bc
54.4 ± 3.8e
EtOH
5.9 ± 0.5bc
36.0 ± 4.6hi
43.1 ± 4.4g
Ginger
MeOH
5.5 ± 1.6bc
70.8 ± 3.3a
75.9 ± 3.9a
EtOH
3.2 ± 0.9ef
54.1 ± 3.4bcd
66.3 ± 5.0bc
Turmeric
MeOH
4.8 ± 0.1cd
51.6 ± 1.6cde
61.4 ± 1.9cd
EtOH
6.2 ± 1.2b
42.7 ± 6.0fgh
46.1 ± 4.0fg
Coriander
MeOH
2.4 ± 0.3f
69.8 ± 1.6a
70.5 ± 2.5ab
EtOH
3.1 ± 0.0ef
49.0 ± 0.5def
51.7 ± 1.6ef
Fennel
MeOH
5.4 ± 0.1bc
46.3 ± 5.2efg
56.2 ± 5.2de
EtOH
4.0 ± 0.0de
34.8 ± 4.7i
41.2 ± 1.2g
Mint
MeOH
3.9 ± 0.1de
59.1 ± 5.7b
62.1 ± 3.2c
EtOH
3.0 ± 0.0ef
40.6 ± 2.9ghi
42.6 ± 1.2g
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05. MeOH = hydromethanolic extracts; EtOH = hydroethanolic extracts; TPC = total phenolic content; DPPH = diphenyl picrylhydrazyl.Significantly higher antioxidant properties were observed in the ginger extract in comparison with extracts of other spices under investigation (Table3). Hydroalcoholic extracts yielded higher free radical scavenging properties with ginger followed by turmeric, i.e., 75.9%, while hydroethanolic extracts presented higher DPPH radical scavenging activities for ginger (66.3%) and coriander (51.7%). Higher DPPH free radical scavenging properties of ginger and turmeric correlates with their higher phenolic contents as compared to the onion extract. DPPH free radical scavenging property of ginger extracts has been previously cited between 67–78% [22]. The considerably higher concentration of hydroxyl rich total phenolics and synergistic role of spices extracts could be achieved by their application as additives in meat and meat-based products.


## 3.3. Antimicrobial Screening of Spice and Herb Extracts

The antimicrobial activity of spice extracts against various pathogenic microbes at 150 ppm concentration is presented in Table4. Inhibition zones of various extracts against Gram-negative and Gram-positive bacteria including E. coli, Salmonella spp., and S. aureus were determined for methanolic and ethanolic extracts of ginger, turmeric, onion, coriander, fennel, and mint extracts at 150 ppm concentration (30–40 μg extracts disc). In comparison with gentamycin and penicillin discs, both methanolic and ethanolic extracts of onion generated larger zones of inhibition against the tested pathogens. Onion extracts generated wider inhibition zones, i.e., 17.1 mm, 16.5 mm, and 15.5 mm, for E. coli, S. aureus, and Salmonella spp., respectively. Comparatively lower antimicrobial activities against tested pathogens were reflected by hydroethanolic extracts of mint, fennel, and coriander extracts.Table 4
Antimicrobial activity of spice extracts against various pathogenic microbes at 150 ppm concentration.
Spices
Extracts
Escherichia coli
Staphylococcus aureus
Salmonella spp.
Gentamycin
—
21.5 ± 0.7a
22.0 ± 0.7a
24.0 ± 0.0a
Penicillin
—
19.5 ± 0.7b
20.5 ± 1.4a
23.5 ± 0.7a
Onion
MeOH
17.1 ± 0.6c
16.3 ± 1.1bc
15.5 ± 3.5b
EtOH
14.5 ± 0.7def
16.5 ± 0.7bc
15.3 ± 0.4b
Ginger
MeOH
11.5 ± 0.7hi
14.5 ± 0.7bcde
9.8 ± 0.4f
EtOH
13.5 ± 2.1efg
16.0 ± 1.4bc
11.0 ± 1.4ef
Turmeric
MeOH
14.8 ± 0.4def
15.5 ± 2.1bc
14.0 ± 1.4bcd
EtOH
16.0 ± 1.4cd
15.0 ± 1.4bcd
15.5 ± 0.7b
Coriander
MeOH
15.2 ± 0.2de
15.5 ± 0.7bc
14.3 ± 0.4bcd
EtOH
13.7 ± 0.2efg
14.0 ± 0.0cde
12.0 ± 0.0def
Fennel
MeOH
13.2 ± 0.3fgh
15.0 ± 0.0bcd
14.8 ± 0.4bc
EtOH
12.3 ± 0.4ghi
12.0 ± 1.4e
13.1 ± 0.2bcde
Mint
MeOH
16.0 ± 0.7cd
16.6 ± 1.3b
14.1 ± 0.4bcd
EtOH
11.0 ± 0.7i
12.7 ± 1.4de
12.7 ± 0.2cde
Mean ± SD; means bearing same letters in a column are statistically nonsignificant atp<0.05. MeOH = hydromethanolic extracts; EtOH = hydroethanolic extracts.


## 3.4. Effect of Extracts’ Supplementation onE. coli Counts in Minced Beef

E. coli counts under refrigeration of minced beef treated with hydromethanolic extracts of spices were estimated in log10 CFU/g during 0–9 days of storage. Interpretation of the data presented in Figure 1 suggests significant (p<0.05) reductions in E. coli counts of minced beef on treatment with varying levels of spice extracts at different storage intervals. In comparison to the negative control where E. coli counts were found to increase from 5.72 log10 CFU/g to 6.29 log10 CFU/g, coriander extracts’ supplementation in E. coli inoculated minced beef presented peak inhibitory properties, i.e., from 4.8 log10 CFU/g to 5 log10 CFU/g, during 9 d refrigerated storage. Around 0.23 log10 CFU/g increase in E. coli counts was observed in minced beef supplemented with 6% ginger extracts as compared to 0.56 log10 CFU/g and 0.60 log10 CFU/g for negative and positive control under similar study conditions. Fennel and mint extracts were also found equally efficacious in inhibiting the pathogenic load of E. coli. Furthermore, methanolic extracts of fennel, mint, and coriander increased the lag period in relation to the normal control. Comparable role of turmeric and onion extracts were noticed against E. coli inhibition. Pearson correlation (r=0.96) shows that the extracts’ amount and storage duration suggested higher E. coli inhibitory properties of spice extracts at an extended amount of supplementation.Figure 1
E. coli inhibitory activity of spice extracts in marinated minced beef under refrigerated (4°C) 9 d storage. NC: negative control, PC: positive control, OM: onion methanolic extracts, GM: ginger methanolic extracts, TM: turmeric methanolic extracts, CM: coriander methanolic extracts, FM: fennel methanolic extracts, and MM: mint methanolic extracts.
(a)
(b)
(c)
(d)
(e)
(f)Antimicrobial activities of spices have been attributed to flavonoids, saponins, glucosinolates, thiosulfinates, and saponins [24]. Ginger bioactive compounds that exhibit antimicrobial activity include ar-curcumin, caryophyllene, ß-sesquiphellandrene, α-farnesene, and zingiberene [25]. Coriander methanolic extracts have been already reported effective against human pathogens including E. coli and Salmonella typhi [26]. An earlier study carried out by Bali et al. [27] endorsed coriander application at the rate of 2–5% in beef sausages to attribute improved meat quality parameters under refrigerated storage for a period of 14 days. The study further suggested coriander application to anticipate ∼1.18 log inhibition of total bacterial count in comparison with normal control during extended refrigerated storage of 21 days. Relatively lesser antibacterial activity of ginger, turmeric, and onion extracts was observed in ground beef that may be associated with poor distribution of spice extracts in a beef matrix. A study conducted by Gupta and Ravishankar [28] revealed that antimicrobial activity of pure pastes of ginger, garlic, and turmeric against E. coli O157:H7 was found higher than that observed in beef, thus suggesting a partial reduction in bactericidal properties of extracts in food system. Ginger extracts have been also reported as proteolytic because they enhance the antimicrobial characteristics against Gram-negative and positive pathogens including E. coli and L. monocytogenes [29].


## 3.5. Effect of Extracts’ Supplementation onS. aureus Counts in Minced Beef

In comparison with both the positive and negative controls, methanolic extracts of tested spices significantly (p<0.05) inhibited S. aureus growth in minced beef during 0–9 days of the study period. Data presented in Figure 2 showed that minced beef marinated with spice extracts at the rate of 1.5–6.0% offered better shelf stability and reduced pathogen growth. Least S. aureus count increment, i.e., 0.24 log10 CFU/g, was observed on 9th day of storage in minced beef marinated with 6% ginger extracts, whereas positive control inoculated with S. aureus at same inoculation levels as of treatment groups was observed with 0.77 log10 CFU/g increase in pathogen counts at the end of the study. Methanolic extracts of coriander and turmeric also delivered pronounced inhibition in S. aureus proliferation during 9 d storage with up to 0.32 and 0.43 log10 CFU/g upsurge for coriander and turmeric extracts, respectively. Fennel, mint, and onion extracts also exhibited significant (p<0.05) S. aureus inhibition in comparison with the normal and positive control (Figure 2).Figure 2
S. aureus inhibitory activity of spice extracts in marinated minced beef under refrigerated (4°C) 9 d storage. NC: negative control, PC: positive control, OM: onion methanolic extracts, GM: ginger methanolic extracts, TM: turmeric methanolic extracts, CM: coriander methanolic extracts, FM: fennel methanolic extracts, and MM: mint methanolic extracts.
(a)
(b)
(c)
(d)
(e)
(f)Ginger extracts have been reported efficacious against both the Gram-positive and Gram-negative bacteria includingS. aureus and E. coli [30]. In an earlier study wherein ginger extracts were applied as natural preservatives in frozen beef sausages, the extracts’ application at the rate of 1.0% was found to increase product shelf stability by significantly inhibiting microbial growth and lipid oxidation [31]. The study in question declares the application of a relatively higher amount of ginger extracts, i.e., 6.0%, to deliver strong microbial inhibitory properties under refrigerated storage. Ginger extracts have also been reported to disrupt and extensively break muscle fibers [32]. This feature enables spice extracts to deliver higher microbicidal activities alongside meat tenderizing properties.The complex composition of minced beef, i.e., carrying a higher amount of lipids, protein, water, and salts, makes it more resistant towards both the synthetic and natural antimicrobials. Hence, higher concentration and amount of spice extracts are desired to offer microbiological inhibitory properties in food in comparison with the microbial growth medium [10]. A study on 43 different spices used in meat-based cuisines of 36 countries concludes that the spices application as food cleanser and their utilization increases in cookeries [33]. Earlier work on spice application in meat-based products suggests palatability of cooked recipes at relatively higher doses of spices than recommended in recent study. Supplementing 10% extracts of myrtle, rosemary, lemon balm, nettle leaves, green tea, and ginger in beef patties, cooked pork meat, and stewed pork had been suggested as organoleptically acceptable [34, 35]. Variability of different spice extracts in presenting microbicidal properties against both E. coli and S. aureus as observed in this study is associated with their varied antimicrobial property-bearing phytochemical profile and growth conditions. Findings from this recent study also suggest relatively higher amount of extract (i.e., 6.0%) supplementation as marinades in meat-based system to offer antimicrobial and other quality enhancement and preservative properties.


## 4. Conclusions

Development of natural ingredient-based blends as preservatives for the meat industry serves emerging challenges including microbiological and oxidative spoilage, pathogenicity, and risks of synthetic additive-associated toxicity. This research demonstrates culinary spices and herbs commonly used in South East Asia and Central Asia as a potential source of antibacterial compounds that might anticipate a broad range of functional and biological properties in meat and meat-based edible goods. This study defines the correlation among phenolic contents and antioxidant activity of spice extracts. However, unique antimicrobial response identified for spices bearing comparatively lower phenolic pool and antioxidant activity is expected to be an outcome of their particular chemical composition with strong bactericidal properties. The findings of this research work are suggestive of spice and herb extracts’ application in beef as culinary agent up to a level of 6% to contribute as a viable strategy in preventing pathogen growth and proliferation under refrigerated storage.



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*Source: 1702086-2019-12-16.xml*