American Journal of Innovative Research and Applied Sciences. ISSN 2429-5396 I www.american-jiras.com
ORIGINAL ARTICLE
MICROBIAL AND PHYSICOCHEMICAL QUALITY OF SOYMILK (GLYCINE MAX) AND TIGER NUT MILK (CYPERUS ESCULENTUS) ENRICHED BY ESSENTIAL OIL FROM CYMBOPOGON CITRATUS (DC) STAPF UNDER ROOM AMBIENT TEMPERATURE IN BURKINA FASO
| Agbémébia Y. AKAKPO 1* | Marius K. SOMDA 1,2 | Donatien KABORE 3 | Henriette B. MIHIN 1 | Abdouramane SAWADOGO 1, Zenabou SEMDE 3 | Fatouma M. ABDOUL-LATIF 4 | Cheik A. T. OUATTARA 1 | and | Aboubakar S. OUATTARA 1 |
1. Départment de Biochimie Microbiologie | Université Joseph KI-ZERBO, Centre de Recherche en Sciences Biologiques Alimentaires et Nutritionnelles (CRSBAN) | Laboratoire de Microbiologie et de Biotechnologies Microbienne (LAMBM) | Ouagadougou, BFA/03 BP : 7021 | Burkina Faso |
2. Départment de Biochimie Microbiologie | Université Joseph KI-ZERBO | Laboratoire de Technologie Alimentaire, Ouagadougou, BFA/03 BP : 7021 | Burkina Faso |
3. Centre National de la Recherche Scientifique et Technologique (CNRST) | Institut de Recherche en Sciences Appliquées et Technologies (IRSAT) | Département Technologies Alimentaires (DTA) | Ouagadougou, BFA/03BP : 7047 | Burkina Faso |
4Institut de Recherches Médicinales | Centre d’Etudes et de Recherche de Djibouti, Route de l’aéroport, BP : 486 | Djibouti, Djibouti |
| Received October 20, 2022 | | Accepted November 04, 2022 | | Published November 12, 2022 | | ID Article | Akakpo-Ref1-6-15ajiras251022 |
ABSTRACT
Background: Tiger nut milk and soymilk produce locally are usually contaminated with spoilage microorganisms and are not storage at ambient temperature. Objective: The present study aims to ensure the preservation of tiger nut milk and soymilk through the antimicrobial properties of the essential oil (EO) of Cymbopogon citratus under ambient temperature condition. Methods: The antibacterial activity was evaluated by agar diffusion and micro-dilution methods. Storage tests were performed under ambient temperature (25 °C) by incorporating tiger nut milk and soymilk with different doses of EO (0.05; 0.10; 0.15; 0.20; 0.25; 0.30; 0.35; 0.40; 0.45 and 0.50 (% v/v)), after which the physicochemical and microbiology characteristics were assessed. Results: The EO showed significant (p ˂ 0.05) inhibitory activity against Bacillus cereus LMG13569 (30.77 mm), Enterococcus faecalis ATCC19433 (22.81 mm), Listeria monocytogenes NCTC9863 (23.63 mm), Staphylococcus aureus ATCC25923 (32.60 mm), Micrococcus luteus LMG3293 (27.84 mm). The results of the shelf-life monitoring showed that the kinds of milk were free of total and thermotolerant coliforms, Bacillus spores and sulphite-reducing anaerobes spores. For the doses of 0.40 to 0.50 % of EO and after 3 days of storage, the total flora load ranged from 1.15x103 to 8.5x102 CFU/ml (0.40 % EO); 1.15x103 to 6.5x102 CFU/ml (0.45 and 0.50 % EO) for the tiger nut milk. The total fungal flora ranged from 9.15x101 to 6.15x101 spores/ml (0.40 % EO); and 9.15x101 to 4.15x101 spores/ml (0.45 and 0.50 % EO). For the soymilk, the total flora load ranged from 1.25x103 to 8.5x102 CFU/ml (0.40 % EO); 1.25x103 to 6.5x102 CFU/ml (0.45 and 0.50 % EO) where the total fungal flora ranged from 102 to 6.15x101 spores/ml (0.40 % EO); 102 to 4.15x101 spores/ml (0.45 and 0.50 % EO). The pH ranged from 6.55 to 6.41 (tiger nut milk) and the acidity value between 0.128 and 0.131% lactic acid (soymilk). Conclusion: The results obtained indicated that the tiger nut milk and soymilk remained physically stable and its microbial quality complied with the standards set for spoilage flora and spores for 3 days under ambient temperature (25 °C). The stabilization concentration can be modulated for sensory acceptability.
Keywords: Essential oil from Cymbopogon citratus, tiger nut milk, soymilk, food spoilage, stabilization, Burkina Faso.
INTRODUCTION
Local beverages are part of the food and cultural use of the population. They are widely consumed in several African countries and attract all age groups [1,2]. They are delicious, nutritious and well appreciated by consumers [1]. Survey data have shown that in recent years, local, plant-based beverages as well as dairy and related products are becoming increasingly important among consumers due to socio-cultural and economic interests in West African countries [3]. In 2017, sales of plant-based beverages represented 127.6 million Euros in France. These new trends can also be seen in other countries such as the United States, where sales of plant-based beverages increased by 9 % in 2017 to 1.4 billion Euros [4]. This new attraction for plant-based beverages comes at the expense of milk. As for milk consumption, it decrease by 6 % in the United States [4] and by 1.6 % in France in 2017 [5]. Among dairy and dairy-like products, soymilk (Glycine max) and tiger nut milk (Cyperus esculentus), are an alternative to cow's milk [6]. They are also used for the formulation of various local dishes and beverages [7,8]. According to surveys conducted in Burkina Faso, soymilk is consumed by 11.91 % of households in Ouagadougou [9]. Soymilk and tiger nut milk as non-alcoholic beverages are nutritional interest and highly recommended for their beneficial effects related to their protein content, quality of fatty acids and content of vitamins, minerals as well as their ability to prevent certain diseases [10,11]. It’s produced in an artisanal way by women who use it as an income generating activity. The production of beverages that heavily involves manual operations without significant pasteurization processes is a source of food pathogen development [12]. These beverages made using local traditional methods and not stabilized offer only a short shelf life due to spoilage microorganisms [13,14]. The short shelf life of tiger nut milk due to spoilage effects by bacteria and molds reduces its consumption [15,16]. The greatest constraint faced by processors is preservation over time [17,18]. Perishable in nature, their preparation, storage, distribution require special attention for protection against spoilage [19] caused by spoilage microorganisms [20]. The natural shelf life of soymilk often does not exceed one week due to several causes namely: failure to follow good hygiene and manufacturing practices, poor pasteurization [18,21]. Food safety is an increasingly important public health concern [22] and also for artisans or agri-food manufacturers [23,24]. Indeed, in the absence of a proper cold chain and the combined effect of temperature, preservation becomes very difficult as conditions become favorable for the development of microorganisms [25,26]. The main concern is undoubtedly microbiological quality, preservation of consumer health, and extension of beverage shelf life [27].
In Burkina Faso, soymilk and tiger nut milk are produced during popular celebrations. The production is mainly artisanal and mostly carried out by women manufacturers who have not received any training in agri-food, often ignoring the Good Hygiene and Manufacturing Practices (GHP/GMP) during the transformation and conservation processes, because, the different source of contamination are the environment, the manufacture, the material, the methods and the product [28,29,30]. However, the milk obtained is a very unstable product, especially in view of the often inappropriate production and marketing conditions. The microorganisms has capable to modify the odor, taste from a load of 106 à 108 CFU/ ml [31]. The increase of the load of bacteria is a potential source of nutritional spoilage [32], acidification and modification of sensory quality [33,34]; the cause of the deterioration of the quality of soymilk produce locally [35]. In response to the lifestyle of consumers who prefer a natural product without synthetic additives with a longer shelf life [36,37], manufacturers have been led to innovate to meet their daily needs in order to ensure marketable quality and reduce economic losses [37]. This situation imposes on the actors of the vegetable milk chain, the development of processing techniques adapted to the socio-economic context [38]. Studies have shown that the major cause of deterioration of local beverages is the action of bacteria, yeasts and molds [32]. In Burkina Faso, studies have shown that local beverages and milk products sold in the streets of Bobo Dioulasso and Ouagadougou are potential sources of pathogenic and spoilage microorganisms, including staphylococci, streptococci, shigella, yeasts and molds [39], Escherichia coli and Salmonella recorded on "zoom koom", "bissap", "gnamakoudji" and ice cream sold in several markets in Ouagadougou [40]. Preservation against spoilage during preparation, storage and distribution is important [23]. To address contamination issues, the rise of chemistry has allowed the use of new substances as synthetic food preservatives. These have been commonly used to prevent food spoilage. The use of chemical preservatives has given satisfaction on inhibiting and/or delaying the development of spoilage bacteria [41]. However, restrictions imposed by international bodies on the use of synthetic preservatives due to their toxicological and carcinogenic effects [42,43,44] [45] has led to a renewed interest in developing more innovative and natural preservation approaches [46,47]. New research has proposed the use of moderate heat treatments combined with natural antimicrobials or natural bioactive molecules for conservation [47,48,49,50].
Some studies have shown that essential oils (EOs) of different plants have an antioxidant, antifungal and antibacterial activities due to their content of bioactive compounds [45,51,52,53]. Their preservative effect has been tested on various food products or in combination with moderate heat treatment methods [54]. The food industry uses EOs to enhance the taste, flavor, and color foods. Among the essential oils, Cymbopogon citratus (C. citratus) EO is of great commercial interest due to its importance in food technology and traditional medicine [55,56,57,58]. Toxicological studies revealed that the most selective substances in C. citratus EO were citralthiosemicarbazone and citral-4-phenyl-3-thiosemicarbazone. At a dose of 2 ml/kg, C. citratus EO did not affect liver and kidney functions [59]. Work has reported that C. citratus EO is less toxic [60]. It has been used as a preservative or alternative to the synthetic preservative approved by Food and Drug Administration (FDA, GRAS, 21 CFR 182.bioactive60) presenting no risk to consumer health according to the 22ième publication of Federal Emergency Management Agency expert panel [61,62] [63]. Its preservative effect has been tested on various food products namely in the stabilization of beer produced in Benin from starch materials against the effects of spoilage due to further fermentation [64], as well as in the preservation of fresh cow's milk [65], milk and fruit juices [66,67]. Other works reported the inhibitory effect of C. citratus EO against molds and such as Aspergillus flavus, Aspergillus parasiticus and Aspergillus ochraceus in yogurt preservation and inhibition of mycotoxin production by these microorganisms [62]. The antibacterial action of the EO of C. citratus has been documented on melon juice through the inhibition of Salmonella enteritidis, Escherichia coli and Listeria [68]. Its preservative effect has been shown on fresh cheese "wara" [63] and cream [69]. The antifungal and antibacterial activities of C. citratus EO have been reported on different microbial species [52-70,71,72,73,74]. The fungicidal effect of C. citratus EO on toxigenic molds found in some foods was reported by Nguefack et al. [75]. Taking into account the biological properties and attributes, C. citratus EO has shown great promise in mitigating the risks of spoilage caused by microorganisms responsible for reduction in marketability [64-66,69,77,78]. It has been used to extend shelf life of foods [76,79]; as a replacement for chemical preservatives [80]. Therefore, the present work was performed to evaluate the ability of C. citratus EO to stabilize tiger nut milk and soymilk under room ambient temperature.
MATERIAL AND METHODS
The essential oil from C. citratus and the tiger nut tubers were used in this study. The extraction of essential oil was carried out by hydrodistillation in adapted Clevenger apparatus from C. citratus leaf dried at room temperature for five days according to the method described by Bayala (2014) [81]. The EO obtained was collected in a sterile amber glass bottle, packed with aluminium and stored at 4 °C in the refrigerator for preservation. The tiger nut tubers and soybean used for the preparation of tiger nut milk and soymilk were purchased from the local market in Ouagadougou (Burkina Faso).
The antibacterial activity study focused on ten (10) strains as indicator microorganisms. The microorganisms used in this study such as Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC9027, Bacillus cereus LMG13569, Bacillus subtilis ATCC6051, Salmonella enteritidis P167807, Yersinia enterocolitica 8A30SKN601, Enterococcus faecalis ATCC19433, Listeria monocytogenes NCTC9863, Micrococcus luteus LMG3293 et Staphylococcus aureus ATCC25923 were purchased from Département Technologie Alimentaire (DTA/IRSAT/CNRST) and Centre de Recherches en Sciences Biologiques Alimentaires et Nutritionnelles (CRSBAN), Université Joseph KI-ZERBO (Burkina Faso).
The tiger nut milk and soymilk were produced following the production technology used by the majority of the producers of local beverages in Burkina Faso with modifications. Briefly, 1 kilogram of each sorted hard tiger nut tubers and soybean was washed in potable water and then washed again with water adding 1 % (w/w) of sodium bicarbonate (NaHCO3) and 1 % (w/w) of salt (NaCl) for 5 minutes. The washed tiger nut tubers and soybean was soaked in 4 liters of drinking water which was been heated to 65 °C and adding sodium bicarbonate 0.2 % (w/w) of the washed tiger nut tubers and soybean for 12 hours. After soaking, the tiger nut tubers and soybean were rinsed with drinking water (65 °C) and then they were ground in a Moulinex blender with 250 g of ice. The mixture obtained was filtered on a muslin cloth with cold water (4 °C). The juice obtained was adjusted to 10 °brix by adding hot water (65 °C) and 1 % (w/v) sugar was added to the different milk obtained. The diagram of the production of tiger nut milk is presented in figure 1.
Figure 1: Diagram of production of unpasteurized tiger nut milk and soymilk.
The effect of essential oil from C. citratus on the stability of tiger nut milk and soymilk was investigated by adding different doses to both tiger nut milk and soymilk. The preservation test was done according to the technique described by Labuza and Schmild (1985) [82]. This technique makes it possible to evaluate the time of alteration at a usual storage temperature for products that have undergone the effect of heat. Thus, a division into several 50 ml batches of each sample of soymilk and tiger nut milk had been carried out, with or without the addition of C. citratus essential oil. The preservation tests of the milk samples were done by incorporating C. citratus EO to the soymilk and tiger nut milk with increasing concentrations of EO. The choice of the doses of EO to be incorporated in the milk was based on the proportion reported by Akakpo et al. (2019) [83]. However, increasing doses of 0.05 - 0.10 - 0.15 - 0.20 - 0.25 - 0.30 - 0.35 - 0.40 - 0.45 and 0.50 % v/v were tested. For each dose of EO the preservation test was done at room temperature (25 °C). Physicochemical and microbiological analyses were determined at 0, 3, 7, 10, 14, 17 and 21 days of storage.
The antioxidant activity of essential oils was determined by the DPPH (2,2-diphenyl -1-picrylhydrazyl) reduction test according to the method described by Burits and Bucar (2000) [84]. This test is based on the reduction of the stable dark purple DPPH (2,2-diphenyl-1-pcrylhydrazil) radical to the reduced yellow DPPH (diphenylpcryl hydrazine) measured at 517 nm with a UV-visible spectrophotometer. Briefly, the methanolic solution of DPPH was obtained by dissolving 4 mg of the powder in 100 ml of methanol. The essential oil samples were prepared in ethanol at different concentrations (2, 4, 6, 8, and 10 µg/ml). A volume of 50 μl of each EO dilution was mixed with 5 ml of 0.004 % (w/v) DPPH methanolic solution. The mixture was incubated for 30 minutes in the dark at room temperature after shaking, and then absorbance was measured at 517 nm against a blank (mixture without essential oil). Ascorbic acid (standards) was prepared according to the same protocol. Measurements were made in quintuplicate and trials were repeated in triplicate. The inhibition rate (% I) of DPPH radicals was expressed as follow:
%
I
=
Ablanc
−
AHE
Ablanc
x
100
% I= {Ablanc -AHE} over {Ablanc} x100
(1)
%I: percentage of DPPH radical’s inhibition;
Ablanc: absorbance of the control reaction (containing the reagents except the essential oil);
AHE: absorbance of the essential oil tested.
The IC50 index, the concentration of antioxidant required to decrease the initial DPPH* concentration by 50 % [85] was obtained by extrapolating to I = 50 % from the inhibition rate curve = f (concentrations). The antioxidant activity index (AAI: Antioxidant Activity Index) was determined by considering the mass of DPPH and the mass of the compound examined in the reaction volume according to the method described by Scherer and Godoy (2009) [86]. The antioxidant activity index (AAI) was calculated as follow:
AAI
=
[
DPPH
]
CI
50
AAI = {left [DPPH right ]} over {CI50}
(2)
[DPPH]: Final concentration of DPPH (µg/ml) in the reaction medium;
IC50: Concentration of antioxidant required to decrease the initial [DPPH*] by 50% (µg/ml).
Preparation of the inocula of fungal strains: The bacterial strains were pre-enriched in nutrient broth. These strains were then plated on nutrient agar at 37°C for 24 hours. From each plate, one colony was selected and then plated on nutrient agar. After incubation, two to three well-isolated colonies obtained from young cultures of the bacterial strains (16 to 18 hours) were picked and dispersed in 10 ml of physiological water (prepared with 0.9 % extra pure NaCl) and adjusted to McFarland standard 0.5 turbidity. The optical density was read at 625 nm. Inocula were adjusted to 108 CFU/ml suspension according to Braga et al. (2007) [87].
Agar diffusion method: Petri dishes containing Mueller-Hinton agar were inoculated aseptically with the inocula. Seeding was done by flooding the Petri dish and the excess was aspirated. After drying the dishes, wells were cut with a sterile cork borer (diameter: 6 mm) in the agar and 7 μl of oil was added to the different wells. The dishes were exposed at room temperature for one (1) hour before incubation to promote the diffusion of the oil on agar plates. The dishes were incubated at 37°C for 24 hours. The presence of a clear zone around the well indicates the inhibition. The results were read by measuring the diameters of inhibition zones (ID) in mm according to Rhayour (2002) [88]. The higher the inhibition diameter, the more sensitive is fungal; but the lowest the inhibition diameter, the more resistant the bacteria [89]. The activity of the EO from C. citratus was determined by measuring the diameter of the inhibition zone appearing around the well. Criteria used by Carovic-Stanko et al. (2010) [90] and Duraffourd et al. [91] were used to evaluate the inhibition diameters (ID) of the essential oil. When ID ˃ 15 mm: the essential oil had high inhibitory action, 10 ≤ ID ≤ 15 mm: the essential oil had moderate inhibitory action and when ID ˂ 10 mm: the essential oil had low inhibitory action [90]. For Duraffourd et al. (1990) [91] when ID ˂ 8 mm: the essential oil is inactive on the bacteria, 8 ≤ ID ≤ 14 mm: the essential oil had intermediary inhibitory action, ID ˃ 20 mm: the essential oil had high inhibitory action.
Determination of minimum inhibitory concentration, minimum fungicidal concentration and minimum bactericidal concentration: Minimum inhibitory concentration and minimum fungicidal concentration were determined by the microdilution method using 96-micro wells [92]. 50 μl Mueller Hinton broth in addition to 0.5 % of tween 80 was inoculated in all the wells of A1 to A11 columns and 100 μl in the wells of A12 column. 50 μl of each oil was added to all the wells of the A1 column. After mixing the contents of wells A1, 50 μl of these wells were used for dilutions in wells A2 to A11. 50 μl of bacteria inocula were inoculated in the wells except for well A11 which contained only the oil and Mueller Hinton broth, and well A12 contained only Mueller Hinton broth. Wells A11 and A12 are the control. The oil concentration used are 500 ; 250 ; 125 ; 62.5 ; 31.25 ; 15.62 ; 7.81 ; 3.91 ; 1.95 and 0.98 μl/ml. The microwell was incubated at 30 °C for 24 hours. After incubation, 100 μl of bacteria suspension subculture from each well of microwell were inoculated on nutritive Agar. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of oil at which no colony was observed after 24 hours of incubation at 37 °C [93]. The bactericidal and bacteriostatic capacity of the oils on bacteria strains were characterized by minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MFC). The ratio MBC/MIC was used to evaluate antibacterial activity. If the ratio MBC/MIC = 1 or 2, the effect is considered as bactericidal; but if the ratio MBC/MIC = 4 or 16, the effect was defined as bacteriostatic [94].
To follow the stability of tiger nut milk and soymilk under room storage conditions, the pH and acidity of the milk were determined. The pH was measured using pH-meter HANNA according to NF T 90-008 [95]. The acidity was determined according to NF EN 12147 V76-130 [96]. The treatments were repeated three times and mean values were calculated. The microbial quality of tiger nut milk and soymilk was appreciated by the numeration of total viable count on Plate Count Agar at 30 °C for 72 hours according to ISO 4833 [97]. The yeasts and mould were enumerated on Sabouraud chloramphenicol Agar at 25 °C for 5 days according to NF ISO 7954 [98]. The Sulfite-reducing anaerobes (SRA) producing spores were searched on liver meat agar at 37 °C for 48 hours according to the method of the standard NF V 08-061 [99]. The count of spore-producing Bacillus was done on bromocresol purple agar (BCP-Glucose) according to the NF V08-407. The total coliform and thermotolerant were numbered according to ISO 4832 [100].
Each measurement was conducted three times. Data were expressed as the mean ± Standard deviation, group means were compared by one-way ANOVA and Tukey test to identify significance (p < 0.05) among groups using the software XLSTAT version 2016.02.27444.
RESULTS
The antioxidant activity of C. citratus EO is presented in table 1. The CI50 value for C. citratus EO was 7.47 µg/ml. The AAI index was 5.3 for C. citratus EO and 7.4 for ascorbic acid at 39.6 µg/ml DPPH. The antioxidant activity index (AAI) of C. citratus EO obtained was lower than that of the control (ascorbic acid) used for all concentrations tested. An inhibition rate of 52.02% for 8 µg/ml of C. citratus EO was recorded. The results obtained showed that the essential oil of C. citratus presented a very strong antioxidant activity evaluated by the antioxidant activity index AAI which was 5.3.
Table 1 : Antioxidant activity index (AAI) value of pure C. citratus EO.
Antioxidant activity index, [DPPH] = 39.6 µg/ml
IC50 (µg/ml)AAI
C. citratus EO7.475.3
Ascorbic acid 5.357.4
[DPPH]: Concentration of Diphenylpicrylhydrazine in the reaction medium; IC50: concentration of antioxidant required to decrease the initial DPPH concentration by 50%; AAI: Antioxidant Activity Index.
The diameters of the inhibition zones of C. citratus EO measured against the tested strains are presented in table 2. The essential oil of C. citratus extracted by hydrodistillation showed antibacterial activity against the tested strains. All tested strains were susceptible. The highly susceptible strains were Bacillus subtilis ATCC6051 and Escherichia coli respectively. The highly susceptible strains were Bacillus cereus LMG13569, Enterococcus faecalis ATCC19433, Listeria monocytogenes NCTC9863, Micrococcus luteus LMG3293 and Staphylococcus aureus ATCC25923, respectively. The largest inhibition diameters were obtained against the bacterial strains namely Bacillus cereus LMG13569 (30.77 ± 0.23 mm), Enterococcus faecalis ATCC19433 (22.81 ± 0.33 mm), Listeria monocytogenes NCTC9863 (23.63 ± 0.30 mm), Staphylococcus aureus ATCC25923 (32.60 ± 0.71 mm), Micrococcus luteus LMG3293 (27.84 ± 0.08 mm). The smallest diameters were obtained against Escherichia coli ATCC25922 (14.89 ± 0.17 mm), Pseudomonas aeruginosa ATCC9027 (10.88 ± 0.59 mm), Salmonella enteritidis P167807 (12.66 ± 0.46 mm) and Yersinia enterocolitica 8A30SKN601 (9.80 ± 0.28 mm). Statistical analyses ANOVA and Tukey's multiple comparison showed a significant difference (p-value ˂ 0.05).
Table 2 : Table presents the diameters of the inhibition zones of C. citratus EO and bacteria strain sensitivity.
Bacteria strainID (mm)Sensitivity Inhibition action
Escherichia coli ATCC25922 Gram-14.89 ± 0.17 hSensitive Moderate inhibitory
Pseudomonas aeruginosa ATCC9027Gram-10.88 ± 0.59 dSensitive Moderate inhibitory
Salmonella enteritidis P167807Gram-12.66 ± 0.46 gSensitive Moderate inhibitory
Yersinia enterocolitica 8A30SKN601Gram-9.80 ± 0.28 dInactive Low inhibitory
Bacillus cereus LMG13569Gram+30.77 ± 0.23 bExtremely sensitiveHigh inhibitory
Bacillus subtilis ATCC6051Gram+20.96 ± 0.20 fHigh sensitiveHigh inhibitory
Enterococcus faecalis ATCC19433Gram+22.81 ± 0.33 eExtremely sensitiveHigh inhibitory
Listeria monocytogenes NCTC9863Gram+23.63 ± 0.30 eExtremely sensitiveHigh inhibitory
Micrococcus luteus LMG3293Gram+27.84 ± 0.08 cExtremely sensitiveHigh inhibitory
Staphylococcus aureus ATCC25923Gram+32.60 ± 0.71 aExtremely sensitiveHigh inhibitory
p-value˂ 0.05
ID: inhibition diameter of essential oil from C. citratus; EO: Essential oil; The values represent the mean of three repetitions ± standard deviation. The same letter (a, b, c, d, e, f, g, h) indicated no statistical difference between the diameters of the inhibition zones in the same column of the strains tested according to ANOVA and Tukey test (p ≥ 0.05).
The minimum inhibitory concentrations and minimum bactericidal concentrations of C. citratus EO measured against the tested strains and the sensitivity are presented in table 3. The results obtained showed that C. citratus EO extracted by hydrodistillation exhibited antimicrobial and antifungal activity on all strains tested with minimum inhibitory concentrations (MICs) ranging from 0.98 to over 250 μl/ml. The minimum bactericidal concentrations (MBCs) recorded for bacterial strains ranged from 3.91 to over 500 μl/ml. Statistical analyses showed a significant difference in MICs between the different microbial strains tested (p-value ˂ 0.05). C. citratus essential oil had a bactericidal effect (BMC/MIC ≤ 2) against Pseudomonas aeruginosa ATCC9027, Listeria monocytogenes NCTC9863, Micrococcus luteus LMG3293 and Staphylococcus aureus ATCC25923. Its effect was bacteriostatic (BMC/MIC ≥ 4) against Escherichia coli ATCC25922, Salmonella enteritidis P167807, Yersinia enterocolitica 8A30SKN601, Bacillus cereus LMG13569, Bacillus subtilis ATCC6051 and Enterococcus faecalis ATCC19433. The low MICs found for C. citratus EO confirm the high activity recorded for the screening test by measuring inhibition diameters.
Table 3 : Table presents the minimum inhibitory and bactericidal concentrations of C. citratus EO and sensitivity of bacteria strain.
Bacteria strainMIC (µl/ml)MBC (µl/ml)MBC/MICSensitivity
Escherichia coli ATCC25922 Gram-1.95 ± 0.00 f7.81 ± 0.00 c4Bacteriostatic
Pseudomonas aeruginosa ATCC9027Gram-7.81 ± 0.00 e31.25 ± 0.00 b2Bactericidal
Salmonella enteritidis P167807Gram-7.81 ± 0.00 e31.25 ± 0.00 b4Bacteriostatic
Yersinia enterocolitica 8A30SKN601Gram-62.50 ± 0.00 c250 ± 0.00 d4Bacteriostatic
Bacillus cereus LMG13569Gram+1.95 ± 0.00 f7.81 ± 0.00 c4Bacteriostatic
Bacillus subtilis ATCC6051Gram+15.62 ± 0.00 d62.50 ± 0.00 e4Bacteriostatic
Enterococcus faecalis ATCC19433Gram+0.98 ± 0.46 a15.62 ± 0.00 f16Bacteriostatic
Listeria monocytogenes NCTC9863Gram+250 ± 0.00 b500 ± 0.00 g2Bactericidal
Micrococcus luteus LMG3293Gram+62.50 ± 0.00 c125 ± 0.00 h2Bactericidal
Staphylococcus aureus ATCC25923Gram+1.95 ± 0.00 a3.91 ± 0.00 a2Bactericidal
p-value˂ 0.05˂ 0.05
EO: Essential oil; MIC: Minimum Inhibitory Concentration of C. citratus essential oil; MBC: Minimum Bactericidal Concentration of C. citratus essential oil. The values represent the mean of three repetitions ± standard deviation. The same letter (a, b, c, d, e, f, g, h) indicated no statistical difference between inhibitory concentration in the same column of the strains tested according to ANOVA and Tukey test (p ≥ 0.05).
The results showed that during storage, the pH value is correlated with the titratable acidity; as the pH decreases, the acidity increases. The results of the monitoring of storage at room temperature (25 °C) showed a stabilization for 3 days (p-value ˂ 0.05) of the pH value between 6.55 and 6.41 and of the acidity between 0.257 and 0.262 % lactic acid of the samples of tiger nuts milk with the doses of 0.45 and 0.50 % EO of C. citratus. Regarding the soymilk samples, it was recorded a stabilization of the acidity value between 0.128 and 0.131 % lactic acid of the soymilk samples for 3 days with the doses of 0.40 to 0.50 % EO of C. citratus (p-value ˂ 0.05). This shows that the tiger nut milk and soymilk samples were stable for 3 days at room temperature with the doses of 0.40 to 0.50 % of C. citratus EO. The pH and acidity of tiger nut milk and soymilk under storage condition are presented in figure 2.
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A1 & B1: pH à 25 °C; A2 & B2: Acidity à 25 °C; LSt: tiger nut milk; LSa: soymilk; EO: Essential oil from C. citratus
Figure 2: Variation of pH and acidity of tiger nuts milk and soymilk under ambient temperature (25 °C).
The results showed that the samples of tiger nuts milk and soymilk under storage at room temperature (25 °C) were free of Bacillus spores and sulfite-reducing anaerobes. An increase in the microbial load of total flora, yeasts and molds of the tiger nuts milk and soymilk samples was recorded with the concentrations of 0 to 0.30 % HE during the 21 days of storage. The samples of tiger nuts milk and soymilk were stable during the first 7 days of storage was obtained with the concentrations of 0.40 to 0.50 % EO of C. citratus. For tiger nuts milk, the total flora load ranged from 1.15x103 to 8.5x102 CFU/ml (0.40 % C. citratus EO); 1.15x103 and 6.5x102 CFU/ml (0.45 % and 0.50 % C. citratus EO). Total fungal flora ranged from 9.15x101 to 6.15x101 spores/ml (0.40 % C. citratus EO); 9.15x101 and 4.15x101 spores/ml (0.45 % and 0.50 % C. citratus EO). For the soymilk samples, total flora load ranged from 1.25x103 to 8.5x102 CFU/ml (0.40 % C. citratus EO); 1.25x103 and 6.5x102 CFU/ml (0.45 % and 0.50 % C. citratus EO). The total fungal flora ranged from 102 to 6.15x101 spores/ml (0.40 % C. citratus EO); 102 and 4.15x101 spores/ml (0.45 % and 0.50 % C. citratus EO). The results of the microbiological analysis of tiger nuts milk and soymilk under storage condition are presented in figures 3 (tiger nut milk) and figure 4 (soymilk).
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Figure 3: Change in the hygienic quality of tiger nuts milk and soymilk under ambient temperature (25 °C). LSt: tiger nut milk; YM: Yeast and Molds; CT: Total coliforms; CTT: Thermotoletants coliforms; ASR: sulphite-reducing anaerobe spores; EO: Essential oil from C. citratus;
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Figure 4: Change in the hygienic quality of soymilk under ambient temperature (25 °C).
LSa: soymilk; YM: Yeast and Molds; CT: Total coliforms; CTT: Thermotoletants coliforms; ASR: sulphite-reducing anaerobe spores; EO: Essential oil from C. citratus.
DISCUSSION
The chemical diversity of the molecules constituting essential oils defines their diverse biological properties. Previous studies have reported a wide variation in the antioxidant activity of C. citratus essential oil. An inhibition rate of 52.02 % for 8 µg/ml EO recorded was higher than that reported by Bayala (2014) [81] for C. citratus EO extracted from leaves collected in Burkina Faso which was 67.58 % for 8 mg/ml EO. The CI50 index of C. citratus EO was 7.47 µg/ml versus 5.35 µg/ml for ascorbic acid. The index antioxidant activity (AAI) was 5.3 and 7.4 respectively for C. citratus EO and ascorbic acid at 39.6 µg/ml DPPH. The variation between AAI can be attributed to the presence of some phenolic and flavonoid compounds in C. citratus EO. Based on the results obtained, C. citratus EO has a very high antioxidant activity (AAI = 5.3) according to the classification of Scherer and Godoy [86]. The EO from C. citratus can replace synthetic antioxidants such as BHA, BHT, and TBHQ that are of concern to consumers due to their safety [101,102]. The antimicrobial activity of EOs is most often correlated with their chemical constituents and functional groups as well as possible synergistic interactions between majority and minority compounds [103,104]. The higher the inhibition diameter, the more sensitive is microorganism, but the lowest the inhibition diameter, resistant is the microorganism [89]. The results obtained showed that following the classification proposed by Duraffourd et al. (1990) [91]; Carovic-Stanko et al. (2010) [90] and Negreiros et al. (2016) [105] all the strains tested were susceptible. Gram positives were more sensitive than Gram negatives. These results are similar to previous work regarding the difference in susceptibility between Gram-positive and Gram-negative strains particularly due to the fact that Gram-positives were more susceptible [103,106]. This is because Gram-negative bacteria have a thin layer of peptidoglycan sandwiched between the plasma membrane and an outer foundation of lipopolysaccharides and proteins. This structure can prevent or protect the peptidoglycan layer from the active ingredient of the oil. The outer membrane of Gram-negative bacteria provides a barrier to the permeability of hydrophobic substances with the ability to cause loss of cytoplasmic components of the cell and bacterial death [107]. Low inhibition diameters were obtained for Yersinia enterocolitica 8A30SKN601 (9.80 ± 0.28 mm) and Pseudomonas aeruginosa ATCC9027 (10.88 ± 0.59 mm) strains. This could be explained by their particular outer membrane structure and their ability to metabolize a range of organic compounds [107,108,109,110]. Bacteria of the genus Pseudomonas are able to utilize terpenes as a source of carbon and energy; they convert limonene (as a model molecule for terpenes) to perillyl alcohol, perillyl acid, aterpineol, or limonene-6,8-diol (Malekey, 2007). C. citratus EO had strong inhibitory activity on Bacillus cereus LMG13569, Enterococcus faecalis ATCC19433, Listeria monocytogenes NCTC9863, Micrococcus luteus LMG3293, and Staphylococcu aureus ATCC25923 strains. Similar results have been reported on Staphylococcus aureus [111], Enterococcus faecalis CIP103907, Listeria monocytogenes CRBIP13.134, Salmonella enterica CIP105150, Salmonella typhimurium ATCC13311, and Shigella dysenteriae 5451CIP [93]. This strong inhibitory activity could be explained by interactions with membrane constituents through the amphipathic nature of the phenolic compounds constituting C. citratus EO [112]. The inhibition diameter with C. citratus EO obtained against Bacillus cereus LMG13569 (30.77 mm) and Staphylococcus aureus ATCC25923 (32.60 mm) was higher than that obtained with the synthetic antibiotic Tetracycline (30 µg) and Ciprofloxacin (5 µg) respectively 19 ± 1.41 mm and 26.5 ± 2.12 mm for Bacillus cereus; 23.5 ± 0.71 mm and 26.5 ± 2.12 mm for Staphylococcus aureus ATCC25923 reported by Semdé et al. (2018) [113]. Similar results were recorded with the synthetic antibiotic Tetracycline (30 µg) and Ciprofloxacin (5 µg) against Escherichia coli ATCC25922 (32.5 ± 3.54 mm and 22.5 ± 0.71 mm), Bacillus subtilis ATCC6051 (30 ± 00 mm and 34 ± 1,41 mm), Yersinia enterocolitica 8A30SKN601 (15.5 ± 0.71 mm and 37 ± 1.41 mm), Salmonella enteritidis P167807 (22.5 ± 2.12 mm and 30.5 ± 2.12 mm) and Pseudomonas aeruginosa ATCC9027 (12 ± 1.41 mm and 32.5 ± 0.71 mm) by Semdé et al. (2018) [113]. For Listeria monocytogenes NCTC9863 (23.63 mm), the inhibition diameter obtained was respectively higher than 21.5 ± 2.12 mm and lower than 31 ± 1.41 mm obtained with the antibiotic Tetracycline (30 µg) and Ciprofloxacin (5 µg) recorded by Semdé et al. (2018) [113]. The high antimicrobial activity results in minimal inhibitory concentrations. However, the minimum effective concentrations must be known, so that the effective antioxidant and/or antimicrobial dose does not exceed organoleptic acceptable levels [114]. For the Escherichia coli strain ATCC25922 a MIC of 1.95 μl/ml was recorded. Previous studies reported MICs respectively lower than 0.5 μl/ml [115] and 9 mg/ml [93]. The MIC found for Pseudomonas aeruginosa ATCC9027 was 1.30 μl/ml. This MIC was lower than 72 mg/ml recorded by Bassolé et al., (2011) [93] and higher than 0.78 mg/ml reported by Nyarko et al. (2012) [116] on the wild type strain. The MIC obtained for Bacillus cereus LMG13569 was 1.95 μl/ml. the work reported by Vyshali et al; (2015) [115] found a MIC of 0.5 μl/ml against Bacillus cereus. For Staphylococcus aureus ATCC25923 the MIC of 1.95 μl/ml found was lower than 0.78 mg/ml recorded by Nyarko et al. [116] against Staphylococcus aureus. However, the MICs found were less than 250 μl/ml, this shows that C. citratus EO has an interesting inhibitory activity against the tested microbial strains [117]. The factors determining the activity of EOs are the chemical composition, the functional groups present in the active components and their synergistic interactions [45,118]. The difference between MICs could be a result of the chemical composition of the EO [93,119]. The results of this study revealed that EO extracted from C. citratus leaves exhibits antioxidant properties, strong antibacterial activity on microbial growth towards the food spoilage and foodborne strains tested and could be used as a natural agent for food preservation [120,121].
During storage at room temperature (25 °C), it was recorded a stabilization for 3 days (p-value ˂ 0.05) with the doses of 0.45 and 0.50 % C. citratus EO of the pH value between 6.55 and 6.41 and of the acidity between 0.257 and 0.262 % lactic acid of the samples of tiger nuts milk. In contrast, the stability of the soymilk samples for 3 days was possible with the doses of 0.40 to 0.50 % C. citratus EO (p-value ˂ 0.05). The results showed that the pH values of the tiger nuts milk and soymilk samples spiked with the concentrations of C. citratus that ensured stability were lower than the pH value of freshly prepared soymilk between 6.5 and 7.71 reported by Iwuoha and Umunnakwe (1997) [122]; Cruz et al. (2007) [123]; Guerrero-Beltran et al. (2009) [124]; Sharma et al. (2009) [125]; Smith et al. (2009) [126] and Tripathi et al. [127]. The pH and titratable acidity values of the soy milk samples were in compliance with the values for cow's milk of 6.5 and 0.15 % lactic acid respectively set by Codex Alimentarius [128]. The results showed that during storage of the samples of tiger nuts milk and soymilk, the pH value was correlated with the titratable acidity; as the pH decreased, the acidity increased. This is explained by the fact that pH is inversely proportional to acidity [129]. Although variations were observed, they were not significant at the 5 % level. In general, the evolution of the results shows a certain stability of the pH and acidity parameters of the samples of tiger nuts milk and soymilk under storage at defined room temperature. The decrease in the pH value of the samples of Tiger Nuts milk and soy milk under storage after a certain time of stability corroborates the results of Khodke et al., (2015) [19] who reported that the pH of stabilized soy milk decreases with increasing storage time. Indeed, plant milks in general are rich media that are subject to a lot of microbial activity [130]. Their storage is often associated with significant microbial contamination, including bacteria and molds [15,16,131,132]. For the tiger nuts milk and soymilk samples, the incorporation of C. citratus EO in the preserved milk also showed a decrease in the microbial load of total flora and yeasts and molds. However, the stability of the tiger nuts milk and soymilk samples confirms the efficacy of the antimicrobial properties of C. citratus EO and the importance of its use in the preservation of Tiger and soy milk. Similar results were reported on the stabilization of yogurt with 0.1 % C. citratus EO for 28 days under storage at 5 °C [62]. Other similar results were reported by Dahouenon-Ahoussi et al., (2012) [64] on the stabilization of beer produced from starch materials against spoilage bacteria due to the effects of fermentation by using 1 ml/L C. citratus EO. The difference could be due to the composition of the food matrix, the type of microorganisms contaminating the products and the initial microbial load. The tiger nuts milk and soymilk under storage at room temperature (25 °C) were free of Bacillus strains, spore-producing sulfito-reducing anaerobes and pathogens; this gives to the tiger nuts milk and soymilk samples a good microbiological and marketable quality. The increase of the load of bacteria is a potential source of nutritional spoilage [32], acidification and modification of sensory quality [33,34]; the cause of the deterioration of the quality of soymilk produce locally [35]. For dairy products, the regulatory shelf life is 15 days under refrigerated conditions at 4 °C, while the use of 0.40 % to 0.50 % EO of C. citratus allowed the storage of tiger nut milk and soymilk samples for 3 days at room temperature (25 °C). For tiger nuts milk samples, total flora load ranged from 1.15x103 to 8.5x102 CFU/ml (0.40% C. citratus EO); 1.15x103 and 6.5x102 CFU/ml (0.45 % and 0.50 % C. citratus EO). Total fungal flora ranged from 9.15x101 to 6.15x101 spores/ml (0.40 % C. citratus EO); 9.15x101 and 4.15x101 spores/ml (0.45 % and 0.50 % C. citratus EO). For the soymilk samples, total flora load ranged from 1.25x103 to 8.5x102 CFU/ml (0.40 % C. citratus EO); 1.25x103 and 6.5x102 CFU/ml (0.45 % and 0.50 % C. citratus EO). The total fungal flora ranged from 102 to 6.15x101 spores/ml (0.40 % C. citratus EO); 102 and 4.15x101 spores/ml (0.45 % and 0.50 % C. citratus EO). The decrease in microbial load of total flora and fungal flora for different increasing concentrations of C. citratus EO corroborates with the idea that C. citratus EO has strong activity on mycelial growth [72,75]. Other similar studies have been reported on the stabilization of cow's milk using C. citratus EO [65]. The tiger nuts milk and soymilk samples spiked with doses greater than 0.40 % of C. citratus were in compliance with the microbiological criteria applicable to plant products and derivatives according to the French legislative and regulatory guide, N°8155 of December 12, 2000 [133], where the tolerance threshold is 103 CFU/g for thermotolerant coliforms (Escherichia coli, 44°C); 102 CFU/g for Staphylococcus aureus and an absence of Salmonella. The microbial load of the total flora of the tiger nuts milk and soymilk samples added with doses higher than 0.40 % EO of C. citratus under storage at room temperature (25 °C) were in conformity with the microbiological criteria set by Soyfoods Association of America [134] and the French legislative and regulatory guide N°8155 of December 12, 2000 [133] which stipulates that the total flora microbial load should be less than 2x105 CFU/ml and 105 CFU/ml respectively. The total flora load of the tiger nuts milk and soymilk with doses higher than 0.40% of C. citratus EO was lower than the microbiological threshold applicable to milk which is 4.70 log germs/ml (5.0119x104 CFU/ml) according to Directive 92/46/CEE [135]. These samples also complied with the microbiological criteria applicable to dairy substitutes as well as pasteurized fruit and vegetable juices, and beverages respectively for total flora 5x105 CFU/ml and for yeasts and molds 102 spores/ml [136].
CONCLUSION
The current study has successfully shown the effectiveness of the essential oils of C. citratus in the stabilization of tiger nut milk and soymilk under room ambient temperature conditions at 25 °C by the proliferation of spoilage flora and yeast and mould. The essential oils of C. citratus can be used as an alternative to the synthetic chemical.
Acknowledgment: This research was supported by the Committee on Scientific and Technological Cooperation of the Organization of Islamic Conference, Islamabad, Pakistan and the International Foundation for Science, Stockholm, Sweden, through a grant to I-3-E-6464-1. The authors also thank the Togolese government for its financial assistance through Direction des Bourses et Stage (DBS-Togo). The authors gratefully acknowledge the donator for their financial support.
Conflict of interest: The present study bears no conflict of interest and I on behalf of my co-authors declare that there is no conflict of interest in submitting this manuscript.
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