Abstract
Background:
The consumption of commercial enteral formulas (CEFs) is increasing worldwide. Given the possibility of contamination of these formulas during preparation and consumption, it is crucial to utilize antimicrobial substances to enhance the safety and quality of them. This study aims to assess the (i) microbiological quality of CEFs and (ii) antibacterial and antioxidant effects of thyme, saffron, and turmeric extract mixture on CEFs.
Methods:
First, 50 samples were collected and analyzed to determine the growth of total viable microorganisms (TVC), coliforms, Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli). Then, the minimum inhibitory concentration of the extracts was determined by the microdilution method, and the antibacterial effects of the mixed extracts were measured on 26 samples by TVC at the time of sampling, 2, 4, and 24 hours after preparation. Additionally, the antioxidant activity of the mixed extracts was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method.
Results:
In the microbial analysis of food formulas, S. aureus and E. coli were not observed, but the coliforms contamination (3.67 ± 0.18 CFU/mL) and TVC (4.29 ± 0.10 CFU/mL) were higher than the standard of U.S. Food and Drug Administration. Additionally, the mixture of aqueous extract of saffron, turmeric, and thyme demonstrated a significant reduction in microbial load within 24 hours. The DPPH analysis indicated that the aqueous extract mixture increased antioxidant activity (38.7%) significantly in comparison to CEFs without extract (19.59%) (P value < 0.001).
Conclusions:
The results of this study indicate that CEFs exhibit high levels of microbial contamination. The extracts of thyme, saffron, and turmeric, however, have the potential to reduce microbial load and enhance antioxidant activity.
Keywords: Antimicrobial activity, antioxidant activity, commercial enteral formula, extract
Introduction
A common problem that many specialists face with hospitalized patients with high metabolic needs is malnutrition.[1,2] Enteral nutrition formulas (ENFs) are mixtures of food and liquid available commercially or handmade. ENFs are used in patients who are malnourished and have an inefficient gastrointestinal system.[3,4] ENFs are used in intensive care units, neonatal intensive care units, and other hospital wards.[5] Ingredients of ENFs include protein sources, dairy products, fruits, and grains. Commercial enteral formulas (CEFs) are types of formulas that are facile to preparation, sterility, energy density, and more nutritious compounds.[6,7] Although CEFs are produced in sterile conditions, due to the appropriate conditions of pH, humidity, nutrients, issues related to contaminated materials, equipment, and improper handling, they are the excellent environment for the growth and proliferation of food-borne microorganisms.[8,9,10]
Although ENFs bypass several of the body’s innate defenses, including low gastric pH, peristalsis, and salivary nitrate circulation, they help protect against microbial overgrowth. In a sense, the lack of food passing through the mouth and the lack of sensory stimuli associated with eating and chewing cause a decrease in peristalsis and a decrease in the production of gastric acid and saliva. The lack of salivary nitrate also reduces the production of nitrite from nitrate by spontaneous anaerobic bacteria on the tongue, which reduces the amount of antimicrobial compounds such as nitric oxide in the stomach.[11] If CEFs are contaminated and the patient’s immune system is disrupted due to chronic disease, the use of drugs, and chemotherapy, there is a possibility of food poisoning and other secondary diseases in patients. Poisoning caused by consumption of formulas causes symptoms such as nausea, vomiting, fever, diarrhea, abdominal discomfort, septicemia, bacteremia, and even death in severe cases.[8,12,13] Common microorganisms found in ENFs include bacteria such as Staphylococcus aureus (S. aureus), Clostridium difficile (C. difficile), coliforms, enterobacteria, Listeria monocytogenes (L. monocytogenes), Salmonella, Escherichia coli (E. coli), and fungi.[3,14] In 2006, the U.S. Food and Drug Administration (FDA) established standards for these foods, particularly for health and nutritional quality, where the amount of aerobic microorganisms in a sample is less than 104 CFU per gram of food (CFU/g). The number of coliforms, salmonella, and L. monocytogenes should not exceed more than 3 colonies per gram of food. The maximum acceptable contamination level for S. aureus is 101 CFU. Also, the total microbial count of ENF samples should be 101 CFU at the beginning of consumption and 103 CFU at the end.[1,3]
Studies conducted in the past have shown that the level of contamination of CEFs is higher than the standard limit, while no strategy has been provided to increase the quality and microbial safety of CEFs.[1,15,16] So, in this study, the antimicrobial activity of natural antimicrobial compounds on CEFs was investigated. Natural antimicrobial compounds such as saffron, turmeric, and thyme can reduce the microbial load and increase the antioxidant activity in foods or formulas.[17,18] Antibacterial properties associated with these plants make them potential preservatives against microbial spoilage and foodborne illness. The mechanism of antimicrobial activity of plants is related to the chemical composition of their extract or essential oil.[17,19] Plant extracts can prevent the growth of pathogenic bacteria and spoilage microorganisms by chemically interfering with the synthesis or function of vital components of the bacterial cell membrane and/or by circumventing conventional antibacterial resistance mechanisms.[20,21] The antimicrobial and antioxidant effects of saffron, turmeric, and thyme extracts have been mentioned in various studies. Therefore, the widespread use of these antimicrobial plants in the forms (powder, extract, and essential oil) in food is very common.[22,23,24,25,26,27,28,29,30]
Therefore, the aim of this study is to evaluate the microbial quality of CEF samples and investigate the effect of the mixture of thyme, saffron, and turmeric extracts on the microbial quality and antioxidant capacity of CEF in three hospitals (a, b, and c) in Isfahan.
Materials and Methods
Extract preparation
To prepare saffron (Isfahan, Iran) extract, 4 g of powdered saffron petals was added to 40 mL of water and the solution was stirred in a shaker for 3 days. The obtained samples were passed through filter paper (Isfahan, Iran) to completely remove suspended particles.[25,31] For turmeric (Isfahan, Iran) extract, 50 g of powder was solved in 500 mL of water. The solution was wrapped with foil and was kept in a dark place for 2 days by shaking on a stirrer. Then it was dried in the freezer for 24 hours. The dried extract was kept in the refrigerator in sterile and dark conditions.[22,32] Thyme extract was also purchased from Zarghani Co., Isfahan, Iran.
Sample preparation
Totally, 76 samples were collected from different ICUs of three hospitals in Isfahan. First, 50 samples were analyzed for microbiological quality, and then another 26 samples were taken to evaluate the antimicrobial and antioxidant effects of extract mixture. CEF solution is prepared several times (once every 2 hours) in the kitchen or intensive care unit of each hospital. 9 g of powder was mixed with 40 mL of water and then poured into the disposable glasses or bottle and consumed. For sampling, 100 mL of feeding solution was transferred to the laboratory under cold and aseptic chain conditions. The entire protocol was approved by the Research Ethics Committee of Isfahan University of Medical Sciences (registration code: IR.MUI.RESEARCH.REC.1401.248).
Microbiological analysis
Samples were tested for TVC, total coliforms, and also for the presence of S. aureus and E. coli using the pure plate technique. A solution of 10 mL of each sample was mixed with 90 mL of sterile normal saline (9 g/L NaCl; Merck, Darmstadt, Germany) and completely homogenized, and then 10-fold serial dilutions of this mixture were prepared. For enumeration of TVC, total coliforms, E. coli, and S. aureus from each dilution, a 1 mL aliquot was added to plate count agar (PCA, Darmstadt, Germany), violet-red bile agar (VRBA, Darmstadt, Germany), and Baird-Parker agar (BPA, Darmstadt, Germany). Colony counts per mL of feed were performed after 24–48 hours of incubation at 37°C. Results are expressed as colony forming units (CFU/mL) of feed. Typical colonies on VRBA and BPA were also examined using appropriate biochemical tests such as Gram stain, catalase test, motility, citrate, indole, and MR/VP tests.[1,33]
Minimum inhibitory concentration and minimum bactericidal concentration
In this study, the antimicrobial activity of extracts from selected plants was evaluated using the microdilution method in 96 multiwell microtiter plates. The microbial strains, S. aureus and E. coli, were cultured on nutrient agar and incubated at 37°C for 24 hours. After this growth period, the bacterial suspensions were standardized with a McFarland scale of 0.5 and then 5 μL of the suspension was diluted to a final concentration of 105 CFU/mL in each well containing Muller Hinton Broth (MHB, Darmstadt, Germany). Equal concentrations of the extracts were prepared, and 50 μL of the stock extracts was added to the wells. The plates were then incubated at 37°C for 24 hours. After incubation, the lowest concentration of the aqueous extract that inhibited the visible growth of the tested microorganisms, indicating the absence of turbidity, was determined as the minimum inhibitory concentration (MIC; μL/mL). In the plate, one row was reserved for the negative control (MHB and extract), another row for the positive control (MHB and bacterial suspension), and the last row for the MHB medium alone.
Muller Hinton Agar (MHA, Darmstadt, Germany) medium was employed to determine the minimum bactericidal concentration (MBC). Five microliters of consecutive dilutions in which no growth was observed were cultured on MHA and incubated for 24 hours at 37°C. Finally, the minimum concentration of the extract at which no colony grew was determined as the MBC.[27,34,35]
Antioxidant activity
The antioxidant activity of the extracts in CEF was determined by the 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay. The CEF was diluted in distilled water to a concentration of 9 g per 40 mL. Subsequently, 800 μL of saffron, 1200 μL of turmeric, and 800 μL of thyme were added to the solution, which was then homogenized for 60 seconds and centrifuged at 2000 rpm for 10 minutes. Subsequently, the clear liquid was diluted at a ratio of 1:10 and employed to assess antioxidant activity. A total of 500 μL of the diluted supernatant was combined with 2.5 mL of a methanolic solution of DPPH (0.1 mM) and incubated for 30 minutes in the dark at room temperature. The absorbance was then measured at 517 nm using a spectrophotometer (BIO and QUEST, England). The percentage of DPPH free radicals was calculated using the following formula:
% Antioxidant activity = ⟦(AA-AB) ÷ AA⟧ × 100
AA is the control absorbance (containing DPPH solution and supernatant without extracts), and AB is the sample absorbance (containing DPPH solution and supernatant with extracts (antioxidant)).[25,36,37]
Antimicrobial activity of extracts
To assess the antimicrobial activity of extracts, 900, 600, and 600 μL of turmeric, saffron, and thyme were added to 30 mL of the original sample solution based on the result of the 96-well plate microdilution method. A solution of 10 mL of each sample containing the mixture of extracts was mixed with 90 mL of sterile normal saline and homogenized completely. Thereafter, 10-fold serial dilutions of this mixture were prepared. For enumeration of TVC, total coliform, E. coli, and S. aureus from each dilution, a 1 mL aliquot was added to the PCA, VRBA, and BPA. For the confirmatory test, biochemical tests such as Gram stain, catalase test, motility, citrate, indole, and MR/VP test were employed.
Statistical analysis
The data were analyzed statistically and descriptively using SPSS software (SPSS, Chicago, IL, USA) version 16. One-way analysis of variance (ANOVA) was employed for the microbial and antioxidant analyses. The antimicrobial effect of the combination of extracts was investigated using the T-test and ANOVA, with the mean ± standard error (SE) employed to determine the difference in the selected times. The significance level for the intended tests was set at 0.05.
Results
Microbiological analysis
The mean CFU/mL of microbial count for CEFs is presented in Table 1. S. aureus and E. coli were not isolated from any of the samples. The mean of TVC (4.29 ± 0.10) and coliforms (3.67 ± 0.18) were higher than the standard in all three hospitals. A total of 47 samples (94%) exhibited a TVC greater than 103 CFU/g, while 36 samples (72%) were contaminated with coliforms. There was no statistically significant difference between hospitals (P value > 0.05). Coliforms and E. coli are indicator microorganisms that are primarily utilized in the assessment of the microbiological quality of food because they are isolated from many food products as food pathogens and have many similarities with other food pathogens.[13,38,39] Some species of coliform isolated from CEFs are shown in Table 2.
Table 1.
Comparison of mean of TVC and coliform of CEFs between three hospitals on preparation time (time 0)
| Colony forming units/mL (cfu/mL)a | Hospital C | Hospital B | Hospital A | Total | P* | ||||
|---|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
||||||
| % | Mean | % | Mean | % | Mean | % | Meanb | ||
| Total viable count | 14/14 (100%) | 4.31±0.04 | 9/9 (100%) | 4.14±0.22 | 27/24 (88.8%) | 4.33±0.18 | 50/47 (94%) | 4.29±0.10 | 0.126 |
| Coliform count | 14/6 (42%) | 3.10±0.26 | 9/6 (66%) | 4.25±0.38 | 27/24 (88.8%) | 3.66±0.23 | 50/36 (72%) | 3.67±0.18 | 0.193 |
aCFU: Colony-forming units. bValues are given as mean±standard error (SE) of two replicates. *Obtained from one-way ANOVA
Table 2.
Type of Enterobacteriaceae detected in CEFs
| Hospital | Type of Enterobacteriaceae | Species | Number of the species |
|---|---|---|---|
| A | Citrobacter | Citrobacter freundii | 4 |
| Serratia | Serratia marcescens | 1 | |
| klebsiella | klebsiella pneumoniae | 2 | |
| klebsiella oxytoca | 2 | ||
| Enterobacter | Enterobacter cloacea | 1 | |
| Enterobacter aerogenes | 1 | ||
| Total | 11 | ||
| B | Citrobacter | Citrobacter freundii | 3 |
| Hafnia | Hafnia alvei | 5 | |
| Shigella | Shigella sonnei | 2 | |
| Total | 10 | ||
| C | Citrobacter | Citrobacter freundii | 3 |
| Yersinia | Yersinia enterocolitica | 1 | |
| Hafnia | Hafnia alvei | 5 | |
| Shigella | Shigella sonnei | 2 | |
| Salmonella | 1 | ||
| Total | 12 |
MIC and MBC determination
The antibacterial activity of aqueous extracts of saffron, turmeric, and thyme was quantified using standard methods. The results demonstrated that these extracts exhibited an inhibitory effect on E. coli and S. aureus but no bactericidal effect. The MICs of saffron, turmeric, and thyme extracts were found to be 100, 100, and 50 μg/mL for E. coli and 50, 100, and 50 μg/mL for S. aureus, respectively.
Antioxidant activity
The antioxidant activity of the combination of aqueous extracts of saffron, turmeric, and thyme was investigated using the DPPH (%) method. As illustrated in Table 3, the mixture of saffron, turmeric, and thyme extracts was able to remove the DPPH radical, with a statistically significant difference observed between samples with extract (38.71 ± 5.94%) and without extract (19.59 ± 3.16%) (P value < 0.05).
Table 3.
Antioxidant activity of combined extract of saffron, turmeric, and thyme by radical scavenging activity DPPH
| Variablea | Mean±SD (%) | Minimum | Maximum | P* |
|---|---|---|---|---|
| CEFb with extract | 38.71±5.94 | 30.27% | 45.82% | <0.001 |
| CEF without extract | 19.59±3.16 | 14.56% | 24.09% | |
| Total | 29.15±10.96 | 14.56% | 45.82% |
aValues are Mean±standard deviation. bCEF: Commercial enteral formulas. *Obtained from one-way ANOVA
Antibacterial activity of extracts
The results of the antimicrobial activity of the mixture of aqueous extract of saffron, turmeric, and thyme in CEFs are presented in Tables 4 and 5. The contamination with TVC and coliforms increased during 24 hours (in periods 0, 2, 4, and 24) in three hospitals, significantly (P value < 0.05). The mixture of extracts was observed to reduce the microbial load of CEFs and to have a preventive and significant effect on the growth of coliform and TVC within 24 hours at intervals of 0, 2, 4, and 24 (P value < 0.05). Although the use of the blend of extracts in hospital A reduced the level of contamination, this reduction was not significant in the studied times (P value > 0.05). Nevertheless, the mixture of extracts demonstrated a significant impact on reducing TVC contamination in hospitals B and C (P value > 0.05). The antimicrobial activity of the blended of extracts in reducing coliform contamination in hospitals A, B, and C was not statistically significant (P value > 0.05) but totally caused a reduction significantly (P value < 0.05) in coliform contamination in comparison to CEFs without extract [Table 5].
Table 4.
Combined effect of saffron, turmeric, and thyme extracts on TVC in CEFs
| Timealine | Time 0 | Time 2 | Time 4 | Time 24 | P* | P** |
|---|---|---|---|---|---|---|
| Total, mean±SEb | ||||||
| Without extract (n=26) | 3.87±0.13 | 4.12±0.12 | 4.39±0.13 | 4.58±0.14 | <0.001 | 0.002 |
| With extract (n=26) | 3.09±0.13 | 3.55±0.12 | 3.84±0.13 | 4.12±0.14 | ||
| P*** | 0.002 | 0.002 | 0.004 | 0.007 | ||
| Hospital A, mean±SE | ||||||
| Without extract (n=12) | 3.57±0.19 | 3.70±0.18 | 3.86±0.16 | 4.03±0.16 | <0.001 | 0.270 |
| With extract (n=12) | 3.06±0.19 | 3.32±0.18 | 3.52±0.16 | 3.76±0.16 | ||
| P | 0.171 | 0.249 | 0.302 | 0.318 | ||
| Hospital B, mean±SE | ||||||
| Without extract (n=6) | 3.77±0.18 | 4.20±0.21 | 4.57±0.25 | 4.72±0.23 | 0.001 | 0.039 |
| With extract (n=6) | 2.22±0.18 | 3.16±0.21 | 3.50±0.25 | 3.83±0.23 | ||
| P | 0.037 | 0.057 | 0.084 | 0.060 | ||
| Hospital C, mean±SE | ||||||
| Without extract (n=8) | 4.38±0.10 | 4.70±0.07 | 5.04±0.08 | 5.30±0.07 | <0.001 | 0.001 |
| With extract (n=8) | 3.78±0.10 | 4.17±0.07 | 4.56±0.08 | 4.87±0.07 | ||
| P | <0.001 | 0.001 | 0.003 | 0.001 |
aTimeline: Time 0: At the time of food preparation, Time 2: 2h after food preparation, Time 4: 4h after food preparation, Time 24: 24h after food preparation. bValue are given mean±standard error (SE) of two replicates. *Obtained from repeated measure ANOVA; P=time. **Obtained from repeated measure ANOVA; P=group (with extract and without extract). ***Obtained from Independent Sample t-test
Table 5.
Combined effect of saffron, turmeric, and thyme extracts on coliforms in CEFs
| Timealine | Time 0 | Time 2 | Time 4 | Time 24 | P* | P** |
|---|---|---|---|---|---|---|
| Total, mean±SEb (CFU/mL) | <0.001 | 0.040 | ||||
| Without extract (n=26) | 2.98±0.21 | 3.83±0.18 | 4.36±0.17 | 4.88±0.16 | ||
| With extract (n=26) | 2.45±0.25 | 3.30±0.21 | 3.89±0.20 | 4.33±0.19 | ||
| P*** | 0.119 | 0.115 | 0.534 | 0.016 | ||
| Hospital A, mean±SE (CFU/mL) | <0.001 | 0.071 | ||||
| Without extract (n=12) | 2.77±0.29 | 3.58±0.17 | 4.01±0.14 | 4.61±0.21 | ||
| With extract (n=12) | 2.42±0.35 | 3.12±0.21 | 3.51±0.18 | 3.96±0.26 | ||
| P | 0.455 | 0.174 | 0.331 | 0.049 | ||
| Hospital B, mean±SE (CFU/mL) | <0.001 | 0.404 | ||||
| Without extract (n=6) | 3.59±0.75 | 4.85±0.51 | 5.13±0.50 | 5.27±0.35 | ||
| With extract (n=6) | 2.71±0.75 | 4.02±0.51 | 4.48±0.50 | 4.92±0.35 | ||
| P | 0.514 | 0.369 | 0.339 | 0.480 | ||
| Hospital C, mean±SE (CFU/mL) | <0.001 | 0.100 | ||||
| Without extract (n=8) | 3.01±0.23 | 3.62±0.24 | 4.55±0.31 | 5.20±0.21 | ||
| With extract (n=8) | 2.25±0.27 | 2.92±0.27 | 4.08±0.36 | 4.47±0.25 | ||
| P | 0.456 | 0.318 | 0.414 | 0.706 |
aTimeline: Time 0: At the time of food preparation, Time 2: 2h after food preparation, Time 4: 4h after food preparation, Time 24: 24h after food preparation. bValue are given mean±standard error (SE) of two replicates. *Obtained from repeated measure ANOVA; P=time. **Obtained from repeated measure ANOVA; P=group (with extract and without extract). ***Obtained from Independent Sample t-test
Discussion
The use of CEFs in hospitals is expanding due to the efficient nutrients and energy and faster preparation. Given the possibility of contamination of CEFs during preparation and consumption, it is crucial to utilize antimicrobial substances to enhance the safety and quality of them. Despite our result, a study revealed and demonstrated that all samples exhibited a TVC below 104 CFU/mL, and no coliforms were detected in CEFs.[33] Additionally, other studies exposed did not observe any TVC or coliforms in CEFs.[13] In the microbial analysis of CEFs, handmade enteral formulas, and infant formulas, all three diets exhibited an acceptable microbial load for patient consumption.[16] However, contamination in CEFs was reported by da Silva Santos et al.[15] A total of 227 samples were collected from hospitals, of which 14 (6.2%) did not comply with the FDA guidelines and exceeded the standard limit for TVC. Additionally, 10 samples had more coliforms than the standard. No Salmonella and S. aureus were isolated from the samples.[40] In another study, 96, 86, and 96% of the 28 CEFs examined were found to be contaminated with TVC, coliforms, and S. aureus, respectively.[1]
The results of this study align with previous studies, indicating the absence of E. coli and S. aureus species. However, the level of contamination with coliforms and TVC is a cause for concern. The presence of high contamination levels indicates a lack of adherence to established hygiene standards during the preparation, storage, and distribution of food. Also, according to FDA standards, some enterobacteriacea exist in samples such as shigella; salmonella showed inefficient hygiene in these foods. This can be attributed to a number of factors, including inadequate temperature and time controls during storage, as well as deficiencies in the manufacturer’s production line, which necessitates a heightened focus on compliance with Hazard Analysis and Critical Control Points (HACCP) principles by personnel involved in the preparation, storage, and distribution of food.[13,15]
The result of antibacterial activity of aqueous extracts of saffron, turmeric, and thyme demonstrated that there is a difference in the sensitivity of the tested bacteria to these extracts. In a study conducted by Jafari-Sales and Pashazadeh,[41] it was demonstrated that the inhibitory effect of the methanolic extract of saffron on S. aureus, B. cereus, E. coli, and P. aeruginosa was 6.25, 12.5, 50, and 100 mg/mL, respectively. In a study conducted by Odo et al.,[42] it was observed that turmeric aqueous extract at concentrations of 0.25 and 0.125 mg/mL did not affect S. aureus and B. cereus. Conversely, in another study, the aqueous extract of turmeric at concentrations of 256, 512, and 1024 μg/mL demonstrated inhibitory effects against methicillin-resistant S. aureus (MRSA), carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Pseudomonas (CRP), and AmpC-producing Enterobacteriaceae.[43] In a study conducted by Gonelimali et al.,[44] it was shown that the ethanol extract of thyme had the most antibacterial activity against Vibrio parahaemolyticus (V. parahaemolyticus), S. aureus, and B. cereus. The aqueous extract of thyme had an inhibitory effect against Salmonella enteritidis (S. enteritidis), in contrast to the ethanolic extract. Also, the aqueous extract showed a significant inhibitory effect on the tested bacteria, with the exception of P. aeruginosa.
The antioxidant activity of the extracts is directly related to the amount of flavonoids and phenolic groups present.[22] In a study conducted by Tanvir et al.,[45] it was shown that aqueous and ethanolic extracts of different turmeric species exhibited DPPH radical inhibition at concentrations of 5.31–16.55 μg/mL and 1.08–3.03 μg/mL, respectively. The ethanol extract had the highest antioxidant activity. Mehrnia et al.[30] reported that the antioxidant activity of thyme, mint, and oregano was 85, 75, and 71%, respectively. Additionally, methanolic and aqueous extracts of saffron revealed IC50 values of 32.16 ± 3.24 and 88.69 ± 6.11, respectively.[25] In all articles, the antioxidant activity of the aqueous extract was found to be lower than that of the methanolic and ethanolic extracts. In the present study, the addition of a combination of saffron, turmeric, and thyme extracts to an amount that does not cause adverse organoleptic changes in the food resulted in a doubling of the antioxidant activity compared to the CEFs without extracts.
Since no studies related to the antimicrobial effect of extracts in CEFs have been conducted so far, we investigated the studies that show the antimicrobial effect of the combination of saffron, turmeric, and thyme extracts in these formulas. The results of the studies show that during the studied period, a significant increase in contamination with TVC and coliforms was observed. Also, the combination of extracts had a significant reduction in the microbial load level of CEFs. However, in hospital A, the level of contamination was lower than in hospitals B and C, although the use of a mixture of extracts has reduced the level of contamination in this hospital, but this reduction was not significant during the study period. In two other hospitals (B and C), the effect of the extract has caused a significant reduction in the amount of contamination of TVC in 24 hours. Also, in general, the amount of coliform contamination in all samples was significant at different times, but due to the reduction in the number of coliforms in CEFs with extracts, this reduction was not significant in any of the three hospitals separately. Similarly, Alsammarraie et al.[46] investigated the antimicrobial activity of turmeric extract coated with silver nanoparticles (AgNPs) on E. coli and L. monocytogenes. They reported that the number of treated cells was significantly lower for both bacteria after 4 hours of incubation with different concentrations. Also, in another study, it was reported that after 12 days of storage at 10°C, the concentration of 200 mg/mL saffron aqueous extract reduced the number of S. Typhimurium by 3.1 ± 0.15 log compared to the control sample; thereby, saffron extract increases shelf life and improves food safety.[47] In the present study, the mixture of extracts was able to reduce the amount of contamination compared to the microbial load observed in the control group. However, by examining the effect of the mixture of extracts in the 24-hour period observed in the intervention group that contained the mixture of extracts, due to the lower level of contamination, the increase in bacterial colony multiplication within 24 hours was more steep. The reason for this increase can be attributed to 1) the competitive growth of other bacteria; 2) the fact that the extracts used were aqueous extracts; naturally, the amount of antimicrobial compounds is lower than alcoholic extracts; and 3) the fact that the stability of the extract decreases over time due to environmental conditions, which can be related to the decrease in the stability of the extract, which requires more studies. So, using extracts in CEFs has a significant impact in preventing food infections in patients and enhancing the quality and durability of it. Nevertheless, when applying plant extracts in a food at an industrial level, it is crucial to consider several key factors, including dosage, effectiveness, mechanism of action, in vitro and in vivo toxicity, and bioavailability tests to gain insight into their behavior in the human body.
Conclusion
The findings of this study indicate that the preparation of CEFs in the three investigated hospitals increases their microbial load beyond the standard level; therefore, it is very important to determine the critical control points in the production, preparation, and distribution of CEFs and running the HACCP system in order to reduce their microbial load. Additionally, the results of this study revealed that the mixture of aqueous extracts of saffron, turmeric, and thyme has the potential to be used as antimicrobial and antioxidant compounds in enteral diet. Although plant extracts are natural compounds and have been included in the list of GRAS compounds by the FDA, standardization and the maximum allowed dose have not been considered for them, which requires further investigation.
Author contributions
RS: Conceptualization, resources, data curation, investigation, methodology, formal analysis, writing – original draft. MS: Writing – review and editing, supervision. MB: Supervision, validation.
Data availability
All data used during this study are included in this published article.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgment
We are grateful to the Student Research Committee, Isfahan University of Medical Sciences for financial support. The present study was carried out with a grant from the Student Research Committee of Isfahan University of Medical Sciences (grant number: 3401412) and ethical code 1401.248.
Funding Statement
Nil.
References
- 1.Baniardalan M, Sabzghabaee AM, Jalali M, Badri S. Bacterial safety of commercial and handmade enteral feeds in an Iranian teaching hospital. Int J Prev Med. 2014;5:604–10. [PMC free article] [PubMed] [Google Scholar]
- 2.Pinto RO, Correia EF, Pereira KC, Costa Sobrinho Pde S, da Silva DF. Microbiological quality and safe handling of enteral diets in a hospital in Minas Gerais, Brazil. Braz J Microbiol. 2015;46:583–9. doi: 10.1590/S1517-838246220131141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Ojo O, Adegboye ARA, Ojo OO, Wang X, Brooke J. The microbial quality and safety of blenderised enteral nutrition formula: A systematic review. Int J Environ Res Public Health. 2020;17:9563. doi: 10.3390/ijerph17249563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Klepper CM, Moore J, Gabel ME, Fleet SE, Kassel R. Pediatric formulas: Categories, composition, and considerations. Nutr Clin Pract. 2023;38:302–17. doi: 10.1002/ncp.10954. [DOI] [PubMed] [Google Scholar]
- 5.Seferoğlu N, Özyürek P, Kısacık ÖG. Enteral nutrition management in the critical care patient: Intensive care nurses'practices of tube feeding. Prog Nutr. 2021;23:e2021263. [Google Scholar]
- 6.Bobo E. Reemergence of blenderized tube feedings: Exploring the evidence. Nutr Clin Pract. 2016;31:730–5. doi: 10.1177/0884533616669703. [DOI] [PubMed] [Google Scholar]
- 7.Milton DL, Johnson TW, Johnson K, Murphy B, Carter H, Hurt RT, et al. Accepted safe food-handling procedures minimizes microbial contamination of home-prepared blenderized tube-feeding. Nutr Clin Pract. 2020;35:479–86. doi: 10.1002/ncp.10450. [DOI] [PubMed] [Google Scholar]
- 8.Bahramian B, Sarabi-Jamab M, Talebi S, Razavi SMA, Rezaie M. Designing blenderized tube feeding diets for children and investigating their physicochemical and microbial properties and dietary inflammatory index. Nutr Clin Pract. 2023;38:360–75. doi: 10.1002/ncp.10893. [DOI] [PubMed] [Google Scholar]
- 9.Bischoff SC, Austin P, Boeykens K, Chourdakis M, Cuerda C, Jonkers-Schuitema C, et al. ESPEN guideline on home enteral nutrition. Clin Nutr. 2020;39:5–22. doi: 10.1016/j.clnu.2019.04.022. [DOI] [PubMed] [Google Scholar]
- 10.Madden AM, Baines S, Bothwell S, Chen E, Goh S, Jerome L, et al. A laboratory-based evaluation of tube blocking and microbial risks associated with one blended enteral feed recipe. J Hum Nutr Diet. 2019;32:667–75. doi: 10.1111/jhn.12685. [DOI] [PubMed] [Google Scholar]
- 11.Smith AR, Macfarlane GT, Reynolds N, O’May GA, Bahrami B, Macfarlane S. Effect of a synbiotic on microbial community structure in a continuous culture model of the gastric microbiota in enteral nutrition patients. FEMS Microbiol Ecol. 2012;80:135–45. doi: 10.1111/j.1574-6941.2011.01279.x. [DOI] [PubMed] [Google Scholar]
- 12.Haughey SA, Chevallier OP, McVey C, Elliott CT. Laboratory investigations into the cause of multiple serious and fatal food poisoning incidents in Uganda during 2019. Food Control. 2021;121:107648. [Google Scholar]
- 13.Vieira MMC, Santos VFN, Bottoni A, Morais TB. Nutritional and microbiological quality of commercial and homemade blenderized whole food enteral diets for home-based enteral nutritional therapy in adults. Clin Nutr. 2018;37:177–81. doi: 10.1016/j.clnu.2016.11.020. [DOI] [PubMed] [Google Scholar]
- 14.Ahangari H, Tarhriz V, Tarighat A, Hashemi M, Ehsani A. Exploring the safety and hygiene of enteral tube feedings by 16S rRNA based sequencing: A risk factor in healthful nutrition. Arch Pharma Pract. 2020;11:129–35. [Google Scholar]
- 15.da Silva Santos AC, Coelho Araújo WM, C de A Akutsu Rde C, Haack de Arruda A. Microbiological quality of enteral feeding and infant formula produced in dietary units, according to the triad of Donabedian. Nutr Hosp. 2015;31:2122–30. doi: 10.3305/nh.2015.31.5.8582. [DOI] [PubMed] [Google Scholar]
- 16.Johnson TW, Milton DL, Johnson K, Carter H, Hurt RT, Mundi MS, et al. Comparison of microbial growth between commercial formula and blenderized food for tube feeding. Nutr Clin Pract. 2019;34:257–63. doi: 10.1002/ncp.10226. [DOI] [PubMed] [Google Scholar]
- 17.Pateiro M, Munekata PES, Sant’Ana AS, Domínguez R, Rodríguez-Lázaro D, Lorenzo JM. Application of essential oils as antimicrobial agents against spoilage and pathogenic microorganisms in meat products. Int J Food Microbiol. 2021;337:108966. doi: 10.1016/j.ijfoodmicro.2020.108966. [DOI] [PubMed] [Google Scholar]
- 18.Stevanović ZD, Bošnjak-Neumüller J, Pajić-Lijaković I, Raj J, Vasiljević M. Essential oils as feed additives—Future perspectives. Molecules. 2018;23:1717. doi: 10.3390/molecules23071717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Vaou N, Stavropoulou E, Voidarou C, Tsigalou C, Bezirtzoglou E. Towards advances in medicinal plant antimicrobial activity: A review study on challenges and future perspectives. Microorganisms. 2021;9:2041. doi: 10.3390/microorganisms9102041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Evelyn, Silva FV. Resistance of Byssochlamys nivea and Neosartorya fischeri mould spores of different age to high pressure thermal processing and thermosonication. J Food Eng. 2017;201:9–16. [Google Scholar]
- 21.Efenberger-Szmechtyk M, Nowak A, Czyzowska A. Plant extracts rich in polyphenols: Antibacterial agents and natural preservatives for meat and meat products. Crit Rev Food Sci Nutr. 2021;61:149–78. doi: 10.1080/10408398.2020.1722060. [DOI] [PubMed] [Google Scholar]
- 22.Akbar A, Khan SA, Ali I. Functional, antioxidant, antimicrobial potential and food safety applications of curcuma longa and cuminum cyminum. Pak J Bot. 2019;51:1129–35. [Google Scholar]
- 23.Akbay GD, Pekcan AG. Turmeric: Evaluation in terms of health and nutrition. J Nutr Diet. 2016;44:68–72. [Google Scholar]
- 24.Amarowicz R, Żegarska Z, Rafałowski R, Pegg RB, Karamać M, Kosińska A. Antioxidant activity and free radical-scavenging capacity of ethanolic extracts of thyme, oregano, and marjoram. Eur J Lipid Sci Technol. 2009;111:1111–7. [Google Scholar]
- 25.Azizian-Shermeh O, Valizadeh M, Taherizadeh M, Beigomi M. Phytochemical investigation and phytosynthesis of eco-friendly stable bioactive gold and silver nanoparticles using petal extract of saffron (Crocus sativus L.). and study of their antimicrobial activities. Appl Nanosci. 2020;10:2907–20. [Google Scholar]
- 26.Bakshi RA, Sodhi NS. Bioactive constituents of saffron plant: Extraction, encapsulation and their food and pharmaceutical applications. Appl Food Res. 2022;2:100076. [Google Scholar]
- 27.de Almeida JM, Crippa BL, de Souza VVMA, Alonso VPP, da Motta Santos E, Júnior, Picone CSF, et al. Antimicrobial action of oregano, thyme, clove, cinnamon and black pepper essential oils free and encapsulated against foodborne pathogens. Food Control. 2023;144:109356. [Google Scholar]
- 28.Hammoudi Halat D, Krayem M, Khaled S, Younes S. A focused insight into thyme: Biological, chemical, and therapeutic properties of an indigenous mediterranean herb. Nutrients. 2022;14:2104. doi: 10.3390/nu14102104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Jayaprakasha GK, Jagan Mohan Rao L, Sakariah KK. Antioxidant activities of curcumin, demethoxycurcumin and bisdemethoxycurcumin. J Agric Food Chem. 2006;98:720–4. doi: 10.1021/jf025506a. [DOI] [PubMed] [Google Scholar]
- 30.Mehrnia MA, Bashti A, Nasab HM. Chemical composition and antioxidant properties of some species of Lamiaceae family from Iran. J Med Plants By-Prod. 2017;6:125–30. [Google Scholar]
- 31.Esmaeelian M, Jahani M, Einafshar S, Fiezy J. Optimization of experimental parameters in subcritical water extraction of bioactive constituents from the saffron (Crocus sativus L.) corm based on response surface methodology. J Food Meas Charact. 2020;14:1822–32. [Google Scholar]
- 32.Tohidi B, Rahimmalek M, Arzani A. Essential oil composition, total phenolic, flavonoid contents, and antioxidant activity of Thymus species collected from different regions of Iran. Food Chem. 2017;220:153–61. doi: 10.1016/j.foodchem.2016.09.203. [DOI] [PubMed] [Google Scholar]
- 33.Moazen M, Rahmdel S, Abdollahzadeh SM, Zahedani MR, Mazloomi SM. Microbiological quality of commercial enteral feedings used in two public hospitals in Shiraz, Iran. J Health Sci Surveill Sys. 2014;2:49–53. [Google Scholar]
- 34.Mostafa AA, Al-Askar AA, Almaary KS, Dawoud TM, Sholkamy EN, Bakri MM. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Saudi J Biol Sci. 2018;25:361–6. doi: 10.1016/j.sjbs.2017.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Zara S, Petretto GL, Mannu A, Zara G, Budroni M, Mannazzu I, et al. Antimicrobial activity and chemical characterization of a non-polar extract of saffron stamens in food matrix. Foods. 2021;10:703. doi: 10.3390/foods10040703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Azadi A, Rafieian F, Sami M, Rezaei A. Investigating the effects of chitosan/tragacanth gum/polyvinyl alcohol composite coating incorporated with cinnamon essential oil nanoemulsion on safety and quality features of chicken breast fillets during storage in the refrigerator. Int J Biol Macromol. 2023;253:126481. doi: 10.1016/j.ijbiomac.2023.126481. [DOI] [PubMed] [Google Scholar]
- 37.Razavi SE, Jafari SM. Effect of corm age on the antioxidant, bactericidal and fungicidal activities of saffron (Crocus sativus L.). stigmas. Food Control. 2021;130:108358. [Google Scholar]
- 38.Metz M, Sheehan J, Feng PCH. Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses-A literature review. Food Microbiol. 2020;85:103283. doi: 10.1016/j.fm.2019.103283. [DOI] [PubMed] [Google Scholar]
- 39.Wen X, Chen F, Lin Y, Zhu H, Yuan F, Kuang D, et al. Microbial indicators and their use for monitoring drinking water quality—A review. Sustainability. 2020;12:2249. [Google Scholar]
- 40.da Silva AC, Rodrigues MX, Silva NCC. Methicillin-resistant Staphylococcus aureus in food and the prevalence in Brazil: A review. Braz J Microbiol. 2020;51:347–56. doi: 10.1007/s42770-019-00168-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Jafari-sales A, Pashazadeh M. Antibacterial effect of methanolic extract of saffron petal (Crocus sativus L.). on some standard gram positive and gram negative pathogenic bacteria in vitro. Curr Perspect Med Aroma Plant (CUPMAP) 2020;3:1–7. [Google Scholar]
- 42.Odo EO, Ikwuegbu JA, Obeagu EI, Chibueze SA, Ochiaka RE. Analysis of the antibacterial effects of turmeric on particular bacteria. Medicine. 2023;102:e36492. doi: 10.1097/MD.0000000000036492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Etemadi S, Barhaghi MHS, Leylabadlo HE, Memar MY, Mohammadi AB, Ghotaslou R. The synergistic effect of turmeric aqueous extract and chitosan against multidrug-resistant bacteria. New Microbes New Infect. 2021;41:100861. doi: 10.1016/j.nmni.2021.100861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Gonelimali FD, Lin J, Miao W, Xuan J, Charles F, Chen M, et al. Antimicrobial properties and mechanism of action of some plant extracts against food pathogens and spoilage microorganisms. Front Microbiol. 2018;9:1639. doi: 10.3389/fmicb.2018.01639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Tanvir EM, Hossen S, Hossain F, Afroz R, Gan SH, Khalil I, et al. Antioxidant properties of popular turmeric (Curcuma longa) varieties from Bangladesh. J Food Qual 2017. 2017:1–8. [Google Scholar]
- 46.Alsammarraie FK, Wang W, Zhou P, Mustapha A, Lin M. Green synthesis of silver nanoparticles using turmeric extracts and investigation of their antibacterial activities. Colloids Surf B Biointerfaces. 2018;171:398–405. doi: 10.1016/j.colsurfb.2018.07.059. [DOI] [PubMed] [Google Scholar]
- 47.Valizadeh S, Moosavy M, Ebrahimi A, Akhondzadeh Basti A, Mahmoudi R, Khatibi S A. The effect of aqueous extract of saffron (Crocus sativus L. Stigma) on the behavior of salmonella typhimurium in a food model during storage at different temperatures. J Nutr Food Sec. 2022;7:37–45. [Google Scholar]
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Data Availability Statement
All data used during this study are included in this published article.