Abstract
The purpose of this study was to determine the effectiveness of the crude extracts in controlling Staphylococcus aureus and Bacillus cereus, as well as the shelf life of the roasted chilli paste. Crude extract was obtained from the volva of Indian oyster mushrooms (Pleurotus pulmonarius) and used to replace preservatives in roasted chilli paste. Thirty isolates of S. aureus and B. cereus were inhibited by crude extract through the disc diffusion method. At a concentration of 2,500 µg/ml, the crude extract inhibited 23 (76.67%) and 20 (66.67%) isolates of S. aureus and B. cereus, respectively, with an inhibition zone size of 7–10 mm. The broth microdilution method was employed in this study to determine the Minimum Inhibitory Concentration (MIC) of the crude extract, and it was observed that the extract at a concentration of 1,000 µg/ml was able to inhibit 18 (60.00%) and 16 (53.00%) isolates of S. aureus and B. cereus, respectively. For the purpose of Minimum Bactericidal Concentration (MBC), the crude extract at a concentration of 1,000 µg/ml was found to kill 16 (53.00%) and 14 (46.00%) isolates of S. aureus and B. cereus, respectively. At a concentration of 4,000 µg/ml, the crude extract had an inhibitory effect from hour 6, lowering S. aureus (Sa16 and Sa21) and B. cereus (Bc5 and Bc12) below the limit of detection (less than 2 log cfu/ml) at 24 h. The ability of the crude extract to inhibit both types of bacteria was observed to be unaffected by heat treatment to a temperature of 65ºC for 30 min (pasteurisation) as well as by no heat treatment at all. The phenolic compounds of the crude extract treated by pasteurisation and without heat treatment were higher than that of the crude extract treated by sterilisation at 121ºC for 15 min. The quality and safety of roasted chilli paste were analysed during storage at 25ºC for 8 months. The quality and safety of roasted chilli paste with crude extract were within the acceptability limit of water activity, total viable count, mould, S. aureus, B. cereus, C. perfringens, and Salmonella spp. as recommended by Thai community product standard. The ability of crude extracts to inhibit S. aureus (Sa16) and B. cereus (Bc5) in roasted chilli paste was then investigated, and it was observed that the crude extract at a concentration of 4,000 µg/ml was able to reduce the S. aureus (Sa16) and B. cereus (Bc5) population to 1.24 and 2.52 log cfu/g, respectively, at 8 months. Therefore, the use of crude extract from Indian oyster mushrooms in conjunction with heat pasteurisation can be employed to improve the shelf life of roasted chilli paste.
Keywords: Food spoilage bacteria, Indian oyster mushroom, Natural food additive, Roasted chilli paste, Shelf life extension
Introduction
Convenience food is one of the most important needs for the present consumers. Consumers are more interested in taking processed foods that are fresh, healthy, and do not contain preservatives such as benzoic acid and sorbic acid. The food preservative standard is that they should not be more than 1,000 ppm [1]. Preservatives should be non-toxic in the body and should not cause illnesses like asthma and cancer [1]. Roasted chilli paste is a processed food item which is made from ingredients such as dried chilli, onion, and garlic. It is flaming and salted with fish sauce, sugar and ripe tamarind. Roasted chilli paste is a popular condiment in Southeast Asia. It is added to food like tom yum or served with toast and sandwiches. The production of roasted chilli paste uses excess Indian oyster mushroom blooms as a protein substitute for dried prawns, thereby increasing the value. Roasted chilli paste has a short shelf life due to the fact that it is made from fresh ingredients which may be moist and contain microorganisms from the soil [2, 3]. The most common contaminants in roasted chilli paste are S. aureus and B. cereus. S. aureus produces enterotoxins that cause gastric and intestinal inflammation. B. cereus produces toxins that cause diarrhoea (diarrhoeal toxin) and vomiting (emetic toxin) [4].
Roasted chilli paste is made with natural extracts instead of preservatives. Indian oyster mushroom (Pleurotus pulmonarius) is one of the most valuable economic crops in the world and is native to India. The mushroom blossoms are off-white to lightly brown and the fibres are quite soft. It has properties that can prevent cancer, lower blood lipids, and act as an antioxidant [3]. An intriguing approach is to use the volva of the Indian oyster mushroom, a waste product, to increase the value of extracting important substances to inhibit the microorganisms in roasted chilli paste. The MIC for Ganoderma lucidum mushroom extract was 8 mg/ml for Escherichia coli and 4 mg/ml for S. aureus. The MBC for both bacteria was also 8 mg/ml [5]. The MIC for Phellinus badius mushroom extract was 35 mg/ml for S. aureus and 10 mg/ml for Listeria monocytogenes [6]. Citric acid extended the shelf life of roasted chilli paste in sterilised retort bags at 121 °C for 20 min. Citric acid was found to be efficient in maintaining the physical and chemical properties of roasted chilli pastes [7]. The essential oils used in roasted chilli pastes were sterilized at 121 °C for 15 min to improve their physicochemical and rheological properties [8]. This work aims to establish the effectiveness of crude extract from Indian oyster mushrooms in inhibiting S. aureus and B. cereus and the possibility of using Indian oyster mushrooms as a biological preservative in extending the shelf life of roasted chilli paste.
Materials and methods
Preliminary analysis of extraction and properties of crude extract from Indian oyster mushroom
In this study, extraction and identification of properties of crude extract from Indian oyster mushrooms were performed at Songkhla Rajabhat University. The Indian oyster mushrooms were washed, baked at 50oC, and then thoroughly crushed to a pulp. The Indian oyster mushroom was extracted with 95% ethanol (Lab-Scan Analytical Sciences, Thailand) in 10 l for 7 days with a 1:2 ratio of Indian oyster mushroom to ethanol (w/v). The ethanol was evaporated using a rotary evaporator (Buchi Rotary Vapour R-114, Switzerland), and the weight of the dried extract was taken. The percentage yield was calculated using the following formula: %yield = weight of mushrooms obtained from extraction/weight of mushrooms used in extraction*100 as modified from [9].
The amount of total phenolic compounds in Indian oyster mushrooms was determined by Folin-Ciocalteu colorimetry. Point two ml of Indian oyster mushroom extract was diluted with 7 ml of distilled water and 5 ml of Folin-Ciocalteu reagent (Merck, Germany), and the mixture was kept in the dark for 4 min, then 15 ml of saturated sodium carbonate solution was added, and the mixture was shaken and kept in the dark for 90 min. The absorbance was read at 760 nm (UV-Vis spectrophotometers, Shimadzu, Japan), comparing to a gallic acid standard. All experiments were carried out in triplicate [10].
The antibacterial activity of a crude extract from Indian oyster mushrooms against S. aureus and B. cereus.
The samples underwent the following analyses:
Determination of the disc diffusion method
S. aureus and B. cereus were isolated from contaminated roasted chilli paste samples. A total of 30 isolates of each species were obtained and subjected to biochemical tests for species-level identification. S. aureus was identified based on Gram-positive cocci morphology, positive catalase, coagulase reactions, mannitol fermentation on Mannitol Salt Agar (MSA; Difco, USA), DNase activity, and a positive Voges-Proskauer (VP) test [11]. In contrast, B. cereus was identified as Gram-positive, rod-shaped, spore-forming, motile bacteria showing β-haemolysis on blood agar, lecithinase activity on Mannitol Egg Yolk Polymyxin Agar (MYP; Difco, USA), positive starch hydrolysis, nitrate reduction, and a positive VP test [12]. The cultures were grown in Tryptic Soy Broth (TSB, Merck, Germany) at 37 °C for 24 h and then subcultured to TSB to reach the concentration of 0.5 McFarland standard, equal to the number of the tested bacteria (1.5 × 108 cfu/ml). The crude extract was dissolved in Dimethyl Sulfoxide (DMSO, Merck, Germany) to a final concentration of 2,500 µg/ml, and 10 µl of the extract was applied to the disc. Thirty isolates each of S. aureus and B. cereus were inoculated in a manner that they covered the entire surface of Muller Hinton Agar (MHA; Merck, Germany). The crude extract disc was placed on the MHA, and the plate was incubated at 37 °C for 18 h. Vancomycin antibiotics (Sigma-Aldrich, Germany) at a concentration of 30 µg/ml were used as a positive control, and the negative control was 10% DMSO. All experiments were carried out in triplicate with some alteration to the method described by [13].
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
S. aureus and B. cereus were sub-cultured in TSB, and the cultures were incubated at 37 °C for 24 h. The cultures were adjusted in TSB to reach the 0.5 McFarland standard, which is approximately 1.5 × 108 cfu/ml of the tested bacteria. The tested bacteria were adjusted in TSB to 5 × 106 cfu/ml; 20 µl was transferred into 160 µl of Muller Hinton Broth (MHB; Merck, Germany) in a microtiter plate (Nunc, Denmark), and then gently mixed to make a final concentration of 5 × 105 cfu/ml. The two-fold dilution of the crude extract was performed in 20 µl, and the concentrations of the crude extract were 1000–2 µg/ml in wells 1–10 while hole 11 had 10% DMSO (without the crude extract). The bacterial inoculum was added to well 12, and the plate was incubated at 37 °C for 18 h. MIC was defined as the lowest concentration of the extract that could prevent the growth of the tested bacteria. Vancomycin antibiotic was used at an 8–0.01 µg/ml concentration as a positive control. All experiments were conducted in triplicate as described by [13]. From the well that gave MIC value, it was spread on Tryptic Soy Agar (TSA, Merck, Germany), and the plate was incubated at 37 °C for 24 h. MBC was defined as the lowest concentration at which the tested bacteria could not grow. All experiments were performed in triplicate as described by [13].
A time-kill assay using the crude extract of Indian oyster mushrooms
There were two isolates of S. aureus and B. cereus that were the most resistant to vancomycin. These were grown in TSB and incubated at 37 °C for 24 h. The cultures were subcultured in TSB to 0.5 McFarland standard or 1.5 × 108 cfu/ml. The tested bacteria were diluted to an initial concentration of 5 × 106 cfu/ml, and 200 µl of the tested bacteria was added to 1,600 µl TSB in the test tube (Pyrex, USA). The final concentration of the tested bacteria was 5 × 105 cfu/ml. Then, 200 µl of the crude extract was added, and the final concentrations of the extract were MIC, 2MIC, and 4MIC. The crude extract was not added to the control group, and the group was incubated at 37 °C for 24 h and then counted every 6 h. The number of tested bacteria on the TSA was counted, and the relationship between log cfu/ml and time (hours) was depicted in the graph (Fig. 1). All experiments were conducted in triplicate [14].
Fig. 1.
Time-kill curve of S. aureus (Sa16) (a), S. aureus (Sa21) (b), B. cereus (Bc5) (c), and B. cereus (Bc12) (d) after treatment with crude extract from Indian oyster mushroom: MIC (♦1,000 µg/ml), 2MIC (▀2,000 µg/ml), 4MIC (▴4,000 µg/ml), and control (✶without crude extract). Data are presented as means ± standard deviation (SD) of three independent experiments (n = 3). Error bars represent SD, but are not clearly visible in the plots due to the relatively small variation (mostly ≤ 0.2 log cfu/ml). The limit of detection was 2 log cfu/ml
Measurement of extravasation content
There were two isolates of S. aureus and B. cereus that were the most resistant to vancomycin. These were grown in TSB, incubated at 37 °C for 24 h, and then centrifuged (Hermle, Germany) at 10,000 g for 4ºC for 5 min. The pellet was suspended in 0.85% normal saline solution (Merck, Germany) to a concentration of 0.5 McFarland standard or 1.5 × 108 cfu/ml and then further diluted to 5 × 105 cfu/ml in 0.85% normal saline solution. Then the crude extract was added at concentrations of 2MIC and 4MIC in a ratio of 1 ml of tested bacteria to 1 ml of crude extract. The inoculum (5 × 105 cfu/ml) was added in this tube (the positive control group), and 10% DMSO was added in this tube (the negative control group). The tubes were then incubated at 37ºC for 0, 6, and 24 h. During the experiment, the samples were centrifuged at 10,000 g for 5 min at 4 ºC. The supernatant was measured at a wavelength of 260 nm. All experiments were performed in triplicate with some alteration to the method described by [15].
Effect of heat on crude extract from Indian oyster mushroom
Crude extracts from Indian oyster mushrooms were heated at 65ºC for 30 min and 121ºC for 15 min. The crude extract was diluted to MIC and 2MIC, and then 200 µl of each concentration were added to tubes containing 1,600 µl of TSB, separately. It was divided into four groups: Group 1 was heated at 65ºC for 30 min (pasteurisation), Group 2 was heated at 121ºC for 15 min (sterilisation), Group 3 was not heated, and Group 4 was the control (each inoculant without crude extract and without heat treatment). Then, 200 µl of each isolate of S. aureus and B. cereus was added to 4 sets of experiments. The final concentration was 5 × 105 cfu/ml and was incubated at 37ºC for 24 h. The tested bacteria were counted with TSA and incubated at 37ºC for 24 h as described by [16]. The amount of phenolic compounds was analysed. All experiments were performed in triplicate as described by [10].
Analysis of the quality and safety of roasted Chilli paste
The roasted chilli paste consisted of 20 g of Indian oyster mushrooms, 21 ml of soybean oil, 15 g of shallots, 13 g of garlic, 11 g of palm sugar, 11 g of ripe tamarind, 5 ml of fish sauce, 3 g of dried red chilli pepper, and 1 g of powdered salt. Indian oyster mushrooms were cut into small pieces. The Indian oyster mushrooms, shallots, garlic, and dried peppers were finely chopped and stirred well. The mixture was blended thoroughly, and palm sugar was added along with fish sauce, salt, ripe tamarind, and vegetable oil. The mixture was then stirred over low heat until cool. The crude extract with a concentration of 4MIC was added to the roasted chilli paste with a ratio of 10 g to 1 ml. The control group did not have crude extracts added. The roasted chilli paste was packaged in a vacuum nylon bag (PA/LLDPE) and should be pasteurised at 65ºC for 30 min. The roasted chilli paste was then soaked in cold water for 10 min. Each group of roasted chilli paste was kept at 25ºC for 8 months. The quality of the roasted chilli paste was analysed every 2 months, while the colour value and proximate composition were analysed during the beginning of production. All experiments were performed in triplicate as described by [17].
The roasted chilli paste sample underwent the following analysis:
Colour-value analysis
The samples were analysed according to L* (lightness), a* (redness), and b* (yellowness) modified from the method of [16].
Water activity analysis (aw)
The sample of aw analysis (Aqualab Lite AL1066, USA) was modified from the method of [18].
Thiobarbituric acid–reactive substances (TBARs)
It was analysed as a 1 g sample with 0.25% Thiobarbituric Acid (TBA; Merck, Germany) and 10% Trichloroacetic Acid (TCA; Merck, Germany) and heated at 95 °C for 15 min. Then centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was used to measure the absorption of light at a wavelength of 532 nm. TBARs (mg/kg) were calculated against the standard malonaldehyde graph modified from the method of [19].
Proximate composition analysis
The sample was analysed for protein, fat, ash, fibre, and carbohydrates [18].
Total plate count
Twenty-five g of sample were transferred to 0.85% normal saline solution (225 ml), and then a 10-fold serial dilution was performed. Total viable count, mould, S. aureus, B. cereus, C. perfringens, and Salmonella spp. were counted with Plate Count Agar (PCA, Difco, USA), Potato Dextrose Agar (PDA, Difco, USA), Baird-Parker Agar (BPA, Difco, USA), Mannitol Egg Yolk Polymyxin Agar (MYP, Difco, USA), Tryptose Sulphite Cycloserine Agar (TSC, Difco, USA), and Xylose Lysine Deoxycholate Agar (XLD, Difco, USA), respectively, and then incubated at 37 °C for 24 h [16]. The standard values of total viable count, mould, S. aureus, B. cereus, Clostridium perfringens, and Salmonella spp. were less than 4, 2, 1, 3, 3, and 0 log cfu/g, respectively [20].
Analysis of the quality and safety of roasted chilli paste after the addition of S. aureus and B. cereus.
An analysis of the quality and safety of roasted chilli paste was carried out when S. aureus and B. cereus were added. A single strain of S. aureus and B. cereus vancomycin-resistant was subcultured in TSB and incubated at 37 °C for 24 h. The cultures were then centrifuged at 10,000 g for 5 min at 4 °C. To the pellets, normal saline solution (supernatant) was added to a concentration of 0.5 McFarland standard, which is equivalent to a concentration of 1.5 × 108 cfu/ml. The solution was then diluted with 0.85% normal saline to achieve a concentration of 5 × 105 cfu/ml. Four groups of roasted chilli pastes were prepared based on the method of [14].
Group 1: To 1000 g of roasted chilli paste was added S. aureus supernatant of 5 × 105 cfu/ml in 100 ml, and 4MIC crude extract in 100 ml. The ratio of roasted chilli paste, S. aureus and crude extract was 1,000 g: 100 ml: 100 ml (10: 1: 1).
Group 2: To 1,000 g of roasted chilli paste was added S. aureus supernatant of 5 × 105 cfu/ml in 100 ml, and 10% DMSO was added. The ratio of roasted chilli paste, S. aureus and 10% DMSO was 1,000 g: 100 ml: 100 ml (10: 1: 1).
Group 3: To 1,000 g of roasted chilli paste was added B. cereus supernatant of 5 × 105 cfu/ml in 100 ml, and 4MIC crude extract was added. The ratio of roasted chilli paste, B. cereus and 4MIC crude extract was 1,000 g: 100 ml: 100 ml (10: 1: 1).
Group 4: In this treatment, to 1,000 g of roasted chilli paste was added B. cereus supernatant of 5 × 105 cfu/ml in 100 ml, and 100 ml of 10% DMSO was added. The ratio of roasted chilli paste, B. cereus and 10% DMSO was 1,000 g: 100 ml: 100 ml (10: 1: 1).
Before the microorganisms were added, the roasted chilli paste was packed in nylon bags (PA/LLDPE) and sterilised at 65 °C for 30 min and soaked in cold water for 10 min. Then, S. aureus and B. cereus cultures were added to the roasted chilli pastes and maintained at room temperature (25 °C). The S. aureus and B. cereus were counted every 2 months for 8 months. All experiments were conducted in triplicate.
Statistical analysis
All experimental data were expressed as mean ± standard deviation (SD) from three independent replicates.
Statistical analysis was performed using one-way analysis of variance (ANOVA) to determine significant differences among treatment groups. Duncan’s new multiple range test (DMRT) was applied as a post hoc test to separate means. All experiments were analyzed using ANOVA, except for the physical and chemical parameters of the roasted chilli pastes, which were compared between the treated and untreated formulas using an independent-samples t-test.
Results
Antibacterial activity of crude extract from Indian oyster mushroom against S. aureus and B. cereus
The crude extract from the cones of the Indian oyster mushroom was sticky, and the colour was light brown. The yield was 19.20%. The total phenolic compound content was 5.12 ± 0.01 mg/g dry weight, and the IC50 was 0.03 ± 0.01 mg/ml.
Disc diffusion method
The outcome of the disc diffusion assay on the ability of crude extract from Indian oyster mushrooms to inhibit S. aureus and B. cereus is shown in Table 1. At 2,500 µg/ml, the crude extract inhibited 23 isolates of S. aureus (76.67%) and 20 isolates of B. cereus (66.67%), with a 7–10 mm inhibition zone. Vancomycin at 30 µg/ml was able to inhibit 17 isolates of S. aureus (56.67%) and 16 isolates of B. cereus (53.33%), with a zone of inhibition of 11–14 mm. Vancomycin at 30 µg/ml was able to inhibit 6 isolates of S. aureus (20.00%) and 10 isolates of B. cereus (33.33%), with a zone of inhibition of 15–18 mm.
Table 1.
The disc diffusion test of crude extract from Indian oyster mushroom against Staphylococcus aureus and Bacillus cereus
| Inhibition zone (mm) | crude extract from Indian oyster mushroom |
Vancomycin | ||
|---|---|---|---|---|
| Number of S. aureus (%) |
Number of B. cereus (%) |
Number of S. aureus (%) |
Number of B. cereus (%) |
|
| 7–10 | 23(76.67) | 20(66.67) | 5(16.67) | 4(13.34) |
| 11–14 | 7(23.33) | 10(33.33) | 17(56.67) | 16(53.33) |
| 15–18 | 0(0.00) | 0(0.00) | 6(20.00) | 10(33.33) |
| 19–22 | 0(0.00) | 0(0.00) | 2(6.66) | 0(0.00) |
Thirty isolates each of S. aureus and B. cereus
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC and MBC results are presented in Table 2. The crude extract from Indian oyster mushrooms at 125-1,000 µg/ml had different effects on S. aureus and B. cereus. Twelve isolates of S. aureus (40.00%) and 20 isolates of B. cereus (66.67%) had MIC values of 1,000 µg/ml. Ten isolates of S. aureus (33.33%) and 19 isolates of B. cereus (63.33%) had MBC values of 1,000 µg/ml. The MIC of vancomycin on S. aureus was 0.12–0.50 µg/ml, and the MBC was 0.50–2.00 µg/ml. The vancomycin antibiotic on B. cereus had an MIC of 0.25–1.00 µg/ml and an MBC of 0.50–4.00 µg/ml (data not shown).
Table 2.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of crude extract from Indian oyster mushroom against Staphylococcus aureus and Bacillus cereus
| Concentration of Crude extract (µg/ml) |
Number of S. aureus (%) | Number of B. cereus (%) | ||
|---|---|---|---|---|
| MIC | MBC | MIC | MBC | |
| 125 | 6(20.00) | 3(10.00) | - | - |
| 250 | 5(16.67) | 3(10.00) | - | - |
| 500 | 7(23.33) | 6(20.00) | 10(33.33) | - |
| 1,000 | 12(40.00) | 10(33.33) | 20(66.67) | 19(63.33) |
Time-kill test
The ability of crude extracts from Indian oyster mushrooms to inhibit S. aureus (Sa16), S. aureus (Sa21), B. cereus (Bc5) and B. cereus (Bc12) for 24 h is shown in Fig. 1. Data are presented as means ± standard deviation (SD) of three independent experiments (n = 3). The MIC and MBC of crude extracts on S. aureus and B. cereus were 1,000 µg/ml. At 2MIC (2,000 µg/ml) and 4MIC (4,000 µg/ml), the crude extracts significantly reduced the number of S. aureus and B. cereus as compared to the initial counts. The number of the tested bacteria began to decrease during hour 6. The best result in inhibiting S. aureus (Sa16) and S. aureus (Sa21) was obtained with 4MIC concentration crude extract. Crude extract at 4MIC reduced the number of S. aureus (Sa16) (5.79 to less than 2 log cfu/ml), and S. aureus (Sa21) (5.75 to less than 2 log cfu/ml) within 24 h (Fig. 1a, b). The 4MIC concentration crude extract reduced the number of B. cereus (Bc5) (5.68 to less than 2 log cfu/ml) and B. cereus (Bc12) (5.70 to less than 2 log cfu/ml) within 24 h (Fig. 1c, d).
Measurement of extravasation content
The measurement of O.D. from Indian oyster mushrooms on S. aureus (Sa16), S. aureus (Sa21), B. cereus (Bc5), and B. cereus (Bc12) over a period of 24 h is shown in Fig. 2. The O.D. of each group at 0 and 24 h was not significantly different (p > 0.001). The O.D. of the crude extracts at 4MIC (4,000 µg/ml) with S. aureus (Sa16) and S. aureus (Sa21) was highest at 6 h. At 4MIC (4,000 µg/ml), the crude extracts had an O.D. of 0.96 for S. aureus (Sa16) and 0.85 for S. aureus (Sa21) (Fig. 2a, b). When mixed with B. cereus (Bc5) and B. cereus (Bc12), the crude extract had an O.D. of 0.90 for B. cereus (Bc5) and 0.85 for B. cereus (Bc12) (Fig. 2c, d).
Fig. 2.
Quantitation of O.D.260 nm of cell constituent release from S. aureus (Sa16) (a), S. aureus (Sa21) (b), B. cereus (Bc5) (c), and B. cereus (Bc12) (d) using crude extract from Indian oyster mushroom at concentrations of 2MIC (2,000 µg/ml; white bars), 4MIC (4,000 µg/ml; horizontal transverse stripe bars) and control group (without crude extract; black bars) at different times. Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test (DMRT). Different lowercase letters above the bars indicate statistically significant differences at p < 0.001
Heat treatment on crude extract from Indian oyster mushroom
The impact of heat on the ability of a crude extract from Indian oyster mushrooms to inhibit S. aureus and B. cereus was shown in Fig. 3. The most effective concentration of the crude extract was 4MIC (4,000 µg/ml). Crude extracts at a concentration of 4MIC (4,000 µg/ml) were pasteurised, and without heat treatment, were capable of reducing S. aureus (Sa16), S. aureus (Sa21), B. cereus (Bc5), and B. cereus (Bc12) to less than 2 log cfu/ml as compared to the control group. In both pasteurization-heated and non-heated crude extracts, the number of S. aureus (Sa16) reduced from 5.75 log cfu/ml to 1.25 and 1.40 log cfu/ml, and the number of S. aureus (Sa21) reduced from 5.70 log cfu/ml to 1.32 and 1.45 log cfu/ml (Fig. 3a, b). Two MIC (2,000 µg/ml) crude extracts were pasteurised-heated and non-heated, and the number of B. cereus (Bc5) was reduced from 5.64 log cfu/ml to 3.12 and 3.10 log cfu/ml, respectively, and the number of B. cereus (Bc12) was reduced from 5.72 log cfu/ml to 3.26 and 3.31 log cfu/ml, respectively (Fig. 3c, d).
Fig. 3.
Crude extracts from Indian oyster mushrooms at concentrations of 2MIC (2,000 µg/ml) and 4MIC (4,000 µg/ml) against inhibition of S. aureus (Sa16) (a), S. aureus (Sa21) (b), B. cereus (Bc5) (c) and B. cereus (Bc12) (d) at 65ºC for 30 min (pasterisation; white bars), 121ºC for 15 min (sterilisation; transverse stripe bars), without heat treatment (polka dot bars), and control group (without extract and without heat treatment; black bars). Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test (DMRT). Different lowercase letters above the bars indicate statistically significant differences at p < 0.001. The limit of detection was 2 log cfu/ml
The effect of heat on the quantity of phenolic compounds in the crude extract of Indian oyster mushroom is shown in Table 3. The phenolic compounds were higher in the pasteurised crude extracts at 2MIC (2,000 µg/ml) and 4MIC (4,000 µg/ml) than in the sterilised crude extracts (p < 0.001). The amount of phenolic compounds in the 4MIC (4,000 µg/ml) crude extract, which was pasteurised without heat treatment, was in the range of 6.17–6.32 mg/g drymass. The phenolic compounds in the 4MIC (4,000 µg/ml) crude extract through sterilisation were in the range of 1.37–1.48 mg/g drymass. The phenolic compounds in the crude extract concentration of 2MIC (2,000 µg/ml) were pasteurised without heat treatment, and the concentration was 5.12–5.23 mg/g drymass. The phenolic compounds in the 2MIC (2,000 µg/ml) crude extract, which were sterilised, were in the range of 1.29–1.35 mg/g drymass.
Table 3.
Effect of heat on the quantity of phenolic compounds in Indian oyster mushroom crude extract
| Tested bacteria | Total phenolic content (mg/g drymass) | ||||||
|---|---|---|---|---|---|---|---|
| Crude extract (2MIC = 2,000 µg/ml) | Crude extract (4MIC = 4,000 µg/ml) | Control | |||||
| T1 | T2 | T3 | T4 | T5 | T6 | ||
| S.aureus(Sa16) | 5.20 ± 0.02B | 1.31 ± 0.05C | 5.12 ± 0.05B | 6.27 ± 0.07A | 1.42 ± 0.12C | 6.21 ± 0.10A | 0.00 ± 0.00D |
| S.aureus (Sa21) | 5.17 ± 0.05B | 1.35 ± 0.04C | 5.20 ± 0.02B | 6.25 ± 0.05A | 1.40 ± 0.09C | 6.30 ± 0.04A | 0.00 ± 0.00D |
| B.cereus(Bc5) | 5.18 ± 0.07B | 1.29 ± 0.02C | 5.15 ± 0.07B | 6.19 ± 0.06A | 1.37 ± 0.04C | 6.17 ± 0.05A | 0.00 ± 0.00D |
| B.cereus (Bc12) | 5.20 ± 0.10B | 1.30 ± 0.05C | 5.23 ± 0.11B | 6.32 ± 0.04A | 1.48 ± 0.03C | 6.25 ± 0.02A | 0.00 ± 0.00D |
Values are expressed as mean ± standard deviation (SD)
Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Duncan’s new multiple range test (DMRT) to determine significant differences among treatment groups
Different superscript uppercase letters (A, B, C, D) within the same row indicate statistically significant differences at p < 0.001
T1 = crude extract (2MIC) + pasteurisation
T2 = crude extract (2MIC) + sterilisation
T3 = crude extract (2MIC) + without heat treatment
T4 = crude extract (4MIC) + pasteurisation
T5 = crude extract (4MIC) + sterilisation
T6 = crude extract (4MIC) + without heat treatment
Control = without crude extract and without heat treatment
Analysis of the quality and safety of roasted Chilli paste
Table 4 shows the physical and chemical values of roasted chilli paste using crude extract from Indian oyster mushrooms at a concentration of 4MIC (4,000 µg/ml) compared to the control group at time 0.
Table 4.
Physical and chemical values of roasted Chilli pastes
| Parameters | Parameter values | |
|---|---|---|
| Treated formula (4MIC = 4,000 µl) | Untreated formula (Control) |
|
| L* | 27.20 ± 1.14 | 27.06 ± 1.30 |
| A* | 14.32 ± 0.09 | 14.10 ± 1.25 |
| B* | 23.27 ± 1.20 | 23.62 ± 1.18 |
| Protein (%) | 12.50 ± 0.35 | 12.65 ± 0.20 |
| Fat (%) | 9.32 ± 0.17 | 9.35 ± 0.25 |
| Ash (%) | 6.34 ± 1.15 | 6.45 ± 0.23 |
| Fiber (%) | 7.48 ± 0.40 | 7.53 ± 0.35 |
| Carbohydrate (%) | 27.18 ± 1.12 | 27.44 ± 1.25 |
Values are expressed as mean ± standard deviation (SD)
Statistical analysis was performed using independent-samples t-test
No statistically significant differences were found between treated and untreated formulas (p > 0.05)
The colour values of the roasted chilli paste containing the crude extract and the control group were not different (p > 0.05). The brightness value (L*) and the value (a*) indicated a reddish and greenish hue, while the value (b*) indicated the yellowness and bluishness of the roasted chilli paste containing the crude extract, with the values 27.20, 14.32, and 23.27, respectively.
The chemical composition did not differ (p > 0.05) between the roasted chilli paste containing the crude extract and the control group. The protein, fat, ash, fibre, and carbohydrate content of the roasted chilli paste containing the crude extract were 12.50, 9.32, 6.34, 7.48, and 27.18%, respectively.
Roasted chilli paste containing crude extract was packed in a nylon bag (polyamide, PA) and kept in vacuum storage at 25 °C, and the quality was evaluated every 2 months until the end of 8 months (Table 5), and the results were as follows:
Table 5.
Chemical and Microbiological values of roasted Chilli pastes in nylon bags, stored at 25ºC for 8 months
| Parameters | Time (months) | Parameter values | |
|---|---|---|---|
| Treated formula (4MIC = 4,000 µl) | Untreated formula (control) | ||
| aw |
0 2 4 6 8 |
0.78 ± 0.12aA 0.80 ± 0.08aA 0.83 ± 0.10aA 0.83 ± 0.05aA 0.85 ± 0.10aA |
0.81 ± 0.02aA 0.84 ± 0.10aA 0.90 ± 0.06aA 0.91 ± 0.07aA 0.96 ± 0.13aA |
| TBARs |
0 2 4 6 8 |
1.25 ± 0.20dA 1.57 ± 0.14dA 2.12 ± 0.18cB 2.34 ± 0.17bB 2.96 ± 0.30aB |
1.20 ± 0.21dA 1.44 ± 0.28dA 3.78 ± 0.15cA 5.12 ± 0.31bA 6.64 ± 0.16aA |
| Total viable count |
0 2 4 6 8 |
1.95 ± 0.12dA 2.54 ± 0.21cB 3.18 ± 0.14bB 3.80 ± 0.10aB 3.92 ± 0.08aB |
2.25 ± 0.09dA 3.40 ± 0.15cA 5.14 ± 0.22bA 6.52 ± 0.26aA 6.65 ± 0.14aA |
| Mould count |
0 2 4 6 8 |
1.00 ± 0.12aA 1.10 ± 0.15aA 1.16 ± 0.08aA 1.37 ± 0.16aB 1.42 ± 0.10aB |
1.00 ± 0.08cA 1.22 ± 0.17cA 1.35 ± 0.06cA 2.36 ± 0.14bA 3.54 ± 0.20aA |
| Staphylococcus aureus count |
0 2 4 6 8 |
0.00 ± 0.00aA 0.00 ± 0.00aA 0.00 ± 0.00aA 0.00 ± 0.00aB 0.00 ± 0.00aB |
0.00 ± 0.00cA 0.00 ± 0.00cA 0.00 ± 0.00cA 2.58 ± 0.12bA 3.86 ± 0.08aA |
| Bacillus cereus count |
0 2 4 6 8 |
0.00 ± 0.00bA 0.00 ± 0.00bA 0.00 ± 0.00bB 1.34 ± 0.15aB 1.47 ± 0.22aB |
0.00 ± 0.00bA 0.00 ± 0.00bA 3.64 ± 0.09aA 3.78 ± 0.12aA 3.96 ± 0.20aA |
| Clostridium perfringens count |
0 2 4 6 8 |
0.00 ± 0.00bA 0.00 ± 0.00bA 0.00 ± 0.00bB 1.27 ± 0.10aB 1.40 ± 0.15aB |
0.00 ± 0.00bA 0.00 ± 0.00bA 3.46 ± 0.30aA 3.62 ± 0.30aA 3.85 ± 0.30aA |
| Salmonella spp. count |
0 2 4 6 8 |
0.00 ± 0.00aA 0.00 ± 0.00aA 0.00 ± 0.00aA 0.00 ± 0.00aB 0.00 ± 0.00aB |
0.00 ± 0.00bA 0.00 ± 0.00bA 0.00 ± 0.00bA 1.14 ± 0.10aA 1.27 ± 0.18aA |
Values are expressed as mean ± standard deviation. Units of microbial counts are expressed as log cfu/g
Units of Thiobarbituric Acid-Reactive Substance (TBARs) are expressed as mg malonaldehyde/kg
Statistical analysis was conducted using one-way analysis of variance (ANOVA) to evaluate significant differences. Duncan’s new multiple range test (DMRT) was used as a post hoc test for multiple comparisons
Different superscript uppercase letters (A, B) in the same row indicate statistically significant differences between treated and untreated formulas (p < 0.001)
Different superscript lowercase letters (a-d) in the same column indicate statistically significant differences among time points for each treatment (p < 0.001)
No statistically significant differences were observed for water activity (p > 0.05)
The water activity (aw) of the roasted chilli paste containing the crude extract and the control group was not different (p > 0.05) at months 0–8. The aw of the roasted chilli paste containing the crude extract was between 0.78 and 0.85, but the aw of the control group was between 0.81 and 0.96. The aw value of the control group at 4–8 months was above the standard value which, is that aw should be less than 0.85.
The rancidity values (TBARs) in roasted chilli paste containing the crude extract and the control group were different (p < 0.001) at months 0–8. The longer the roasted chilli paste was stored, the higher the TBARs value. The TBARs of the roasted chilli paste containing the crude extract were stored for 4, 6, and 8 months, and the results were 2.12, 2.34, and 3.16 mg malonaldehyde/kg, respectively. The TBARs of the control group were 3.78, 5.12, and 6.64 mg malonaldehyde/kg, respectively.
The total viable count was different (p < 0.001) in the roasted chilli paste containing the crude extract and the control group at months 2–8. There was an increase in the total viable count with the storage of the roasted chilli paste. The total viable count in the roasted chilli paste containing the crude extract at months 2, 4, 6, and 8 was 2.54, 3.18, 3.80, and 3.92 log cfu/g, respectively. The total viable count in the control group at 4–8 months was higher than the required limit of less than 4 log cfu/g [20].
The amount of mould in the roasted chilli paste containing the crude extract and the control group had different values (p < 0.001). The amount of mould in the roasted chilli paste containing the crude extract at months 6 and 8 was1.37 and 1.42 log cfu/g, respectively. The mould count in the control group of roasted chilli paste at 6–8 months was higher than the standard of 2 log cfu/g [20].
B. cereus and C. perfringens were counted, and there was a good repetition of the results, and the levels of B. cereus and C. perfringens in the roasted chilli paste containing the crude extract and the control group were different (p < 0.001). At months 6–8, B. cereus and C. perfringens in roasted chilli pastes with the crude extract were less than 2 log cfu/g. At months 4–8, the concentration of B. cereus and C. perfringens in the control group was higher than the required levels of less than 3 and 3 log cfu/g, respectively [20]. S. aureus and Salmonella spp. were not detected in the roasted chilli pastes with the crude extracts at months 0–8. At months 6–8, S. aureus and Salmonella spp. in the control group were below the detection limit of 1 log cfu/g, and Salmonella spp. was not detected in 25 g of roasted chilli paste.
The roasted chilli paste containing the crude extract at a concentration of 4MIC (4,000 µg/ml), S. aureus (Sa16), and B. cereus (Bc5) was packed in a vacuum nylon bag and stored at 25 °C. The frequency of S. aureus (Sa16) and B. cereus (Bc5) was determined every 2 months until the end of 8 months (Table 6). The number of S. aureus (Sa16) and B. cereus (Bc5) in the roasted chilli paste containing the crude extract and the control group was different at 2–8 months (p < 0.001). After 8 months of storage, the number of S. aureus (Sa16) decreased from 5.72 log cfu/g to 1.24 log cfu/g in the roasted chilli paste containing the crude extract. The level of S. aureus more than 1 log cfu/g. The number of B. cereus (Bc5) was reduced from 5.75 log cfu/g to 2.52 log cfu/g in roasted chilli paste containing crude extract for 8 months. The level of B. cereus was less than 3 log cfu/g. The control group of roasted chilli pastes was superior to S. aureus and B. cereus throughout the 8-month storage periods.
Table 6.
Microbiological values of roasted Chilli pastes inoculated with Staphylococcus aureus (Sa16) and Bacillus cereus (Bc5) packed in nylon bags, stored at 25ºC for 8 months
| Tested bacteria | Time (months) | Plate count (log cfu/g) | |
|---|---|---|---|
| Treated formula (4MIC = 4,000 µl) | Untreated formula (control) | ||
| S. aureus (Sa16) |
0 2 4 6 8 |
5.72 ± 0.09aA 3.25 ± 0.06bB 2.45 ± 0.20cB 1.18 ± 0.10dB 1.24 ± 0.15dB |
5.70 ± 0.14aA 5.46 ± 0.18aA 5.32 ± 0.26aA 4.43 ± 0.17bA 4.26 ± 0.13bA |
| B. cereus (Bc5) |
0 2 4 6 8 |
5.75 ± 0.12aA 3.70 ± 0.15bB 2.84 ± 0.22cB 2.61 ± 0.08cB 2.52 ± 0.10cB |
5.82 ± 0.17aA 5.53 ± 0.20aA 5.47 ± 0.27aA 4.58 ± 0.19bA 4.50 ± 0.21bA |
Values are expressed as mean ± standard deviation (SD)
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s new multiple range test (DMRT) to determine significant differences
Different superscript uppercase letters (A, B) in the same row indicate statistically significant differences between treated and untreated formulas at p < 0.001
Different superscript lowercase letters (a-d) in the same column indicate statistically significant differences among time points for each tested bacteria at p < 0.001
Discussion
The cone of Indian oyster mushroom was extracted using 95% ethanol, and the crude extract had a yield of 19.20%. Phenolic compounds were quantified at 5.12 mg/g dry weight, and the IC50 value was 0.03 mg/ml. The IC50 value of the crude extract was relatively low, which indicated that it had high antioxidant activity. A previous study reported that Craterellus cornucopioides (black trumpet mushroom) had an IC50 value between 0.06 and 0.1 mg/ml. Each of the plants had different levels of phenolic compounds; therefore, it had different antioxidant activity [21].
The disc diffusion method was used as a first test to find Indian oyster mushroom extracts that inhibited S. aureus and B. cereus. The ability of the crude extracts to inhibit S. aureus and B. cereus was assessed by applying crude extracts on MHA. The crude extracts had an MIC value of 1,000 µg/ml against large groups of S. aureus and an MBC value of the same amount for B. cereus. Crude extracts obtained using different solvents had different amounts of phenolic compounds, and had different antioxidant activities. Different levels of inhibition were caused by the source of mushroom extraction, the presence or absence of solvent polarity, and the strain of the microorganism [22]. The Pleurotus ostreatus extract was tested to see how well it worked against S. aureus and E. coli. The MIC values were 2 and 2 µg/ml, and the MBC values were 4 and 4 µg/ml [23]. With the time kill method, the crude extracts were tested on S. aureus and B. cereus. At hour 6, the crude extracts lowered the number of S. aureus and B. cereus. S. aureus and B. cereus were both inhibited by the crude extract at a concentration of 2MIC (2000 µg/ml). Their counts were reduced by about 4 and 2 log cfu/g, respectively, compared to the initial number of the tested bacteria. The crude extract at a concentration of 2MIC (2000 µg/ml) inhibited S. aureus and B. cereus and reduced their counts by approximately 4 and 2 log cfu/g, respectively, from the initial number of the tested bacteria. At concentration of 4MIC (4000 µg/ml) of the crude extract, S. aureus and B. cereus were inhibited, and the counts were reduced below the limit of detection (less than 2 log cfu/g) at 24 h. Phenolic compounds, found in mushrooms, such as flavonoids, hydroxybenzoic acid, hydroxycinnamic acid, and phenolic acid have antimicrobial, anticancer, and anti-inflammatory activity [22].
The effects of the crude extract on S. aureus and B. cereus at different time periods revealed that the crude extract at a concentration of 4MIC (4000 µg/ml) had a higher O.D. than the crude extract at a concentration of 2MIC (2000 µg/ml) at hour 6. The increase in O.D. at 260 nm was due to the permeabilisation of the cytoplasmic membrane by phenolic compounds, which led to the release of low-molecular-weight metabolites: nucleotides, purines, pyrimidines, and amino acids. It affected the process of metabolism and thereby killed the microorganism. The decrease in O.D. after 24 h was caused by biomolecules (DNA and RNA) breaking down, cell stopping to divide or entry into the stationary phase, and the killing of the cells [3].
The studies on the heat treatments on the activity of crude extracts established that the crude extracts at a concentration of 2MIC (2,000 µg/ml) through pasteurisation and without heat treatment reduced the number of S. aureus and B. cereus by 4 and 2 log cfu/ml, respectively, when compared with the initial tested bacteria. The crude extract at a concentration of 4MIC (4,000 µg/ml) was subjected to pasteurisation and without heat treatment could inhibit S. aureus and B. cereus. However, crude extracts subjected to sterilisation could not inhibit S. aureus and B. cereus. Therefore, the phenolic compounds in the crude extracts were higher at concentrations of 2MIC (2,000 µg/ml) and 4MIC (4,000 µg/ml) through pasteurisation and without heat treatment than in the crude extracts that were subjected to sterilisation. The sterilisation process altered or degraded the phenolic compounds in the crude extract. The degradation of phenolic compounds when exposed to heat was a complex process that involved several mechanisms, such as oxidation, structural changes, and reduction in antioxidant activity [22]. For example, temperatures higher than 100 °C caused the phenolic compounds in chilli shrimp paste to degrade compared to 80 °C for 21.6 min [16].
The analysis of colour and chemical composition of roasted chilli paste in crude extract at a concentration of 4MIC (4,000 µg/ml) and the control group (without crude extract) was not different. The raw materials used in the production of roasted chilli pastes were essentially the same. The a* value was low and the b* value was high because it contained sugar, ripe tamarind, and garlic. The brightness value (L*) was low because the raw material was dark. Stir frying and sterilising the roasted chilli paste at 65 °C for 30 min resulted in the release of fluid that covered the surface of the roasted chilli paste [8]. Previous research showed that adding lime essential oil to habanero chilli paste maintained the colour quality; however, the heat reduced the colour quality [8]. However, a study on citric acid at 0.5% in chilli pastes sterilised at 121 °C for 15 min had values for L*, a*, and b* in the chilli paste of 39.84, 13.51, and 28.45, respectively [7]. The roasted chilli paste was analysed for its chemical composition, and it was revealed that the test groups and the control group were not different. Indian oyster mushrooms were the most important ingredient added to the roasted chilli paste, and their proportion was 20% of the total weight. Therefore, carbohydrates were the highest, and proteins were the second. Indian oyster mushrooms are a high-protein plant which can be used to replace some proteins in roasted chilli pastes such as dried shrimp. This makes it possible for those who are allergic to shrimp to be able to use chilli paste [24]. Therefore, adding and not adding crude extract to roasted chilli paste did not alter the colour or chemical composition.
The water activity (aw) value indicates the minimum amount of water in the food in which the microorganism can grow and cause various chemical reactions. The aw value changes with the humidity, and a low aw reading indicates that the food contains low moisture [8]. The aw of the roasted chilli paste (test groups) was lower than 0.85, while the control group was higher than 0.85 at month 4. The aw value of roasted chilli paste with crude extract was less than 0.85 according to the community product standard [20].
TBARs analysis is done to quantify the production of reactive oxidative species as a measure of lipid oxidation (rancidity). The TBARs values of the test groups and control group were different after 4 months. The TBAR values of the test groups were less than 3, while the control group was greater than 3 from month 4 onwards. The TBAR values of greater than 3 mean that the food has started to develop a bad smell. The TBAR values of greater than 7 indicate that the food has a different smell and is spoilt. The long storage period led to an increase in TBARs and thus the formation of a rancid taste. Accordingly, the TBARs of meat curry treated at 121 °C for 7 min increased with increased storage time [25]. Dried chilli fish paste with sodium benzoate added causes an increased TBARs value when stored for a longer period [26].
Crude extract was added to roasted chilli paste and was kept at 25 °C for 8 months. The tested group was found that the total viable count, mould, B. cereus, and C. perfringens did not exceed the standard value, and S. aureus and Salmonella spp. were not found. The total viable count, B. cereus, C. perfringens were analysed in the control group at months 4–8, and mould, S. aureus, and Salmonella spp. were analysed in the control group at months 6–8, which is above the community product standard [20]. Chan et al. [27] reported that ginger extract added to roasted chilli paste could best control Staphylococcus spp. The total viable count was reduced by 3 log cfu/g, and the mould count was reduced by 4 log cfu/g over the 5-day storage period.
Researchers looked at how the crude extracts of Indian oyster mushrooms affected the growth of S. aureus and B. cereus in roasted chilli paste. The number of bacteria decreased in both the test and control groups after 8 months of storage at 25 °C. The test groups were better at inhibiting both types of tested bacteria than the control group. This was because the phenolic compounds broke through the cytoplasmic membrane, stopped the infection ‘s metabolism, and leading to death [3]. The inhibition was due to components in the roasted chilli paste, such as garlic, which had allicin; shallots, which had sulphide; and chilli pepper, which had capsaicinoids. Panang curry contains chilli, lemongrass, shallot, garlic and spices and for that reason had good inhibition against S. enteritica and L.monocytogenes [28].
The addition and non-addition of the crude extract to the roasted chilli paste had no effect on the aw change. It was possible to meet the community product standard for 8 months of storage at 25 °C because the crude extract was added to the roasted chilli paste. This reduced the number of TBARs, total viable counts, mould, S. aureus, B. cereus, C. perfringens, and Salmonella spp. The crude extract was effective in inhibiting S. aureus and B. cereus that were incorporated into the roasted chilli paste and stored at 25ºC for 8 months. The use of crude extract from Indian oyster mushrooms instead of preservatives in roasted chilli pastes, which were packed in nylon bags (PA/LLDPE) and pasteurised was an example of applying hurdle technology. The roasted chilli paste was stored at 25ºC and had an appropriate value. General stores such as shopping malls could sell them because roasted chilli paste prevented the tested bacteria from multiplying [29].
Conclusion
The crude extract from Indian oyster mushroom had a yield of 19.20%, phenolic compounds of 5.12 mg/g dry weight, and an IC50 of 0.03 mg/ml. A lot of S. aureus and B. cereus could not stand up to the crude extract, which had MIC and MBC values of 1000 and 1000 µg/ml, respectively. The crude extract at a concentration of 4MIC (4,000 µg/ml) inhibited both tested bacteria and disrupted the cytoplasmic membrane at 6 h. A crude extract at a concentration of 4MIC (4,000 µg/ml) was pasteurised (30 min, 65ºC) and without heat treatment and inhibited both tested bacteria. For 15 min at 121ºC, the crude extract did not inhibit either of the bacteria that were tested. The amount of phenolic compounds in the crude extract at a concentration of 4MIC (4,000 µg/ml), which was pasteurised and without heat treatment, was higher than that of the crude extract which was sterilised. The roasted chilli paste with the crude extract was kept at 25ºC for 8 months and met the community product standard in aw, TBARS, total viable count, mould, S. aureus, B. cereus, C. perfringens, and Salmonella spp. results. S. aureus and B. cereus were inoculated into the roasted chilli paste. The results of the studies indicated that the crude extract decreased S. aureus and B. cereus during the storage period at 25ºC for 8 months. Therefore, this hurdle technology (pasteurisation combined with crude extract) was effective in meeting the community product standard for 8 months.
Acknowledgements
The authors would like to thank the Faculty of Science and Technology, Songkhla Rajabhat University, for providing the funding. We acknowledge David James Sims for assistance with manuscript preparation.
Author contributions
Atchara Nuphet Phoem is responsible for conceptualisation, methodology, writing, review, and original draft preparation. Rawita Osit is responsible for the experiment and the original draft preparation. All authors have read and agreed to the published version of the manuscript.
Declarations
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Dey S, Nagababu BH (2022) Applications of food color and bio-preservatives in the food and its effect on the human health. Food Chem Adv 1:100019. 10.1016/j.focha.2022.100019 [Google Scholar]
- 2.Srilarp J, Ongkunaruk P (2020) The study of a small-sized Thai chilli paste supply chain. In IOP conference Series: Materials Science and Engineering, 773(1): 012014. IOP Publishing. 10.1088/1757-899X/773/1/012014
- 3.Yadav D, Negi PS (2021) Bioactive components of mushrooms: processing effects and health benefits. Int Food Res 148:110599. 10.1016/j.foodres.2021.110599 [DOI] [PubMed] [Google Scholar]
- 4.Bintsis T (2017) Foodborne pathogens. Aims Microbiol 3(3):529–563. 10.3934/microbiol.2017.3.529 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Erbiai EH, Amina B, Kaoutar A et al (2023) Chemical characterization and evaluation of antimicrobial properties of the wild medicinal mushroom ganoderma lucidum growing in Northern Moroccan forests. Life 13(5):1217. 10.3390/life13051217 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Vazquez-Armenta FJ, Leyva JM, Mata-Haro V et al (2022) Phenolic compounds of Phellinus spp. With antibacterial and antiviral activities. Braz J Microbiol 53(3):1187–1197. 10.1007/s42770-022-00745-x [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Pangastuti HA, Permana L, Fitriani V, Mareta DT, Wahyuningtyas A (2020) The effect of citric acid on chemical and physical characteristics of andaliman (Zanthoxylum acanthopodium) chilli paste in retort packaging. In IOP Conference Series: Earth and Environmental Science, 537(1), 012031. IOP Publishing. 10.1088/1755-1315/537/1/012031
- 8.Medina-Torres N, Cuevas-Bernardino JC, Ayora-Talavera T et al (2021) Changes in the physicochemical, rheological, biological, and sensorial properties of Habanero Chilli pastes affected by ripening stage, natural preservative and thermal processing. Rev Mex Ing Quim 20(1):195–212. 10.24275/rmiq/Alim1768 [Google Scholar]
- 9.Phoem AN, Voravuthikunchai SP (2012) Growth stimulation/inhibition effect of medicinal plants on human intestinal microbiota. Food Sci Biotechnol 21:739–745. 10.1007/s10068-012-0096-z [Google Scholar]
- 10.Heleno SA, Barros L, Martins A et al (2015) Nutritional value, bioactive compounds, antimicrobial activity and bioaccessibility studies with wild edible mushrooms. LWT-Food Sci Technol 63(2):799–806. 10.1016/j.lwt.2015.04.028 [Google Scholar]
- 11.U.S. Food and Drug Administration (FDA) (2020) Bacteriological Analytical Manual (BAM), Chap. 14: Bacillus cereus
- 12.U.S. Food and Drug Administration (FDA) (2016) Bacteriological Analytical Manual (BAM), Chap. 12: Staphylococcus aureus
- 13.CLSI (2024) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically (12th ed.). CLSI standard M07. Clinical and Laboratory Standards Institute
- 14.Niu C, Xue Y, Liu C et al (2020) Identification of gas-forming spoilage bacteria in Chilli sauce and its control using Nisin and salt. LWT 118:108658. 10.1016/j.lwt.2019.108658 [Google Scholar]
- 15.Parichanon P, Sattayakhom A, Matan N, Matan N (2022) Antimicrobial activity of lime oil in the vapour phase against Listeria monocytogenes on ready-to-eat salad during cold storage and its possible mode of action. Food Control 132:108486. 10.1016/j.foodcont.2021.108486 [Google Scholar]
- 16.Sobhi B, Noranizan M, Karim SA et al (2012) Microbial and quality attributes of thermally processed Chilli shrimp paste. Int Food Res J 19(4):1705–1712. http://www.ifrj.upm.edu.my [Google Scholar]
- 17.Cheok CY, Sobhi B, Adzahan NM et al (2017) Physicochemical properties and volatile profile of Chilli shrimp paste as affected by irradiation and heat. Food Chem 216:10–18. 10.1016/j.foodchem.2016.08.011 [DOI] [PubMed] [Google Scholar]
- 18.AOAC International (2019) Official methods of analysis of AOAC International (21st ed.), Chap. 1. AOAC International
- 19.Tajik H, Farhangfar A, Moradi M et al (2014) Effectiveness of clove essential oil and grape seed extract combination on microbial and lipid oxidation characteristics of Raw Buffalo patty during storage at abuse refrigeration temperature. JFPP 38(1):31–38. 10.1111/j.1745-4549.2012.00736.x [Google Scholar]
- 20.Office of industrial product standard (2023) Thai community product standard (Namphrix Phao). 9 pp. https://tcps.tisi.go.th/public/StandardList.aspx
- 21.Kosanić M, Ranković B, Stanojković T et al (2019) Craterellus cornucopioides edible mushroom as source of biologically active compounds. Nat Prod Commun 14(5):1934578–19843610. 10.1177/1934578X19843610 [Google Scholar]
- 22.Rashmi HB, Negi PS (2020) Phenolic acids from vegetables: A review on processing stability and health benefits. Int Food Res 136:109298. 10.1016/j.foodres.2020.109298 [DOI] [PubMed] [Google Scholar]
- 23.Gangwar R, Salem MM, Maurya VK et al (2024) Exploring time-killing and biofilm Inhibition potential of bioactive proteins extracted from two varieties of Pleurotus ostreatus. Front Microbiol 15:1456358. 10.3389/fmicb.2024.1456358 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Srikram A, Supapvanich S (2016) Proximate compositions and bioactive compounds of edible wild and cultivated mushrooms from Northeast Thailand. Anres 50(6):432–436. 10.1016/j.anres.2016.08.001 [Google Scholar]
- 25.Shah MA, Bosco SJD, Mir SA, Sunooj KV (2017) Evaluation of shelf life of retort pouch packaged Rogan Josh, a traditional meat curry of Kashmir, India. Food Packag Shelf Life 12:76–82. 10.1016/j.fpsl.2017.04.001 [Google Scholar]
- 26.Pongsetkul J, Benjakul S (2021) The use of sodium benzoate on shelf-life and quality attributes of dried Chilli fish paste stored in different packaging containers. Foods 10(8):1802. 10.3390/foods10081802 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Chan MT, Nor-Khaizura MAR, Mahyudin NA et al (2021) Antibacterial activity of black Cumin, clove, and ginger essential oils against specific spoilage organisms of ready-to-eat Chilli shrimp paste. Int Food Res J 28(2):393–400. 10.47836/ifrj.28.2.21 [Google Scholar]
- 28.Sapabguy C, Yasurin P (2015) Natural antibacterial activity of Thai red curry paste in coconut milk based curry; Kang-Kati, model on Salmonella Sp. and Listeria monocytogenes. Walailak J Sci Tech 12(5):473–480. 10.14456/WJST.2015.82 [Google Scholar]
- 29.Abdullahi N, Dandago MA (2021) Hurdle technology:principles and recent applications in foods. IFNP 17(1):6–11. 10.22146/ifnp.52552 [Google Scholar]