Shelf-Life Feasibility Study of Moringa oleifera Seasoned Beef

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Introduction
Beef constitutes a vital component of food and dietary requirements in most developing countries [1] Due to the increase in population, the demand for beef is on the rise [1].Unfortunately, raw beef can easily perish if not handled well.For any food product, food safety is a top priority for the producers before the sensory quality.Te capacity to keep food-borne microbes and pathogenic organisms below regulatory limits is crucial for extending the shelf life of beef because microorganisms are one of the signifcant variables that afect the stability of meat [2].Studies have indicated that the bacterial load of raw meat in Ghana is very high due to the various contamination processes it undergoes from the slaughterhouse to the butcheries [3].Contamination can lead to spoilage of beef and cause food-related illnesses.
Te stability and shelf life of beef is ultimately infuenced by the production process.For example, meat from the conventional market production system tends to have fewer food-borne pathogens than meat from the niche market production system [4].Apart from preslaughter handling which may infuence glycogen levels and hence pH of meat, spoilage of beef may occur as a result of postslaughter activities such as microbial contamination, lipid oxidation, and autolysis [5].Factors, such as the physiological state of the animal at the time of slaughtering, the microbial contamination in the areas of handling, the hygiene practices of the handlers, and the level of contamination of the tools used, may all contribute to the initial microbial load on meat [6] and hence its fnal stability.
Although raw beef is readily available to the wealthy and middle-class consumers in major cities, the majority of impoverished people may not have the means of keeping it fresh.Terefore, many cannot easily access beef due to stability challenges.To keep raw beef fresh, it needs to be frozen.However, due to power instability in most developing countries [7], there is a need to fnd alternative ways of keeping beef fresh.Artifcial preservatives have been used over decades to keep beef stable for a long time.However, these have been associated with health problems, such as cancer, mental retardation, nausea, weakness, and headaches [8].Also, synthetic preservatives tend to reduce the nutritional quality of meat [9].For these reasons, consumers prefer naturally preserved meat products with prolonged shelf life.Te alternatives to increasing the nutritional value and stability of foods against the use of artifcial preservatives are plant-based extracts.Tere have been studies using plant-based extracts to increase the shelf life of meat products [10][11][12][13][14].One such plant extract which has no known negative side efects is Moringa oleifera [15][16][17][18].
M. oleifera is efective against a wide range of foodspoilage bacteria and fungi.It has been reported that Moringa leaves extracts with its reasonable safety margins may be used as an antimicrobial agent to inhibit bacterial growth in food [19].Tis includes those that cause gastric ulcers and gastric cancer.M. oleifera leaf extracts can reduce the microbial load of treated meat under cold conditions [20] and may also act as a natural antioxidant to inhibit lipid oxidation in some meat products [21].Apart from this, it also contains phenolic compounds which possess antiinfammatory, antioxidant, neuroprotective, and hepatoprotective activities [22] which can reduce the risk of cardiovascular diseases and diabetes [23].
Although there are a lot of factors that have an impact on the stability and shelf life of beef, preservation methods that can ensure the quality and stability of meat may contribute to the reduction of food insecurity.Terefore, the objective of this study was to determine the stability of beef treated with M. oleifera leaf extract as an alternative preservation method.

Sample Collection.
Fresh beef of 2 kg was acquired from the Makola market in the Greater Accra region in duplicate.Te meat was aseptically transported to the Microbiology Laboratory at a cold condition of 4 °C.Tis was washed twice under running water and deboned.All connective tissues and fats were detached and the beef was frozen at −18 °C until usage.Certifed leaves of M. oleifera were obtained from an indigenous farmstead in Kasoa in the Central Region of Ghana.Tese were aseptically washed and dried in a Genius food dehydrator Ksh 6500 at 30 °C.

Moringa oleifera Extraction with Ethanol.
Te dried M. oleifera leaves were blended using an electric grinder (Binatone BLG-450 MKS) into a fne powder (100 g), dissolved in 500 ml of ethanol, and incubated at room temperature for 72 hours with stirring at regular intervals.Te extracts were fltered through Whatman No. 41 flter paper and the fltrates were vacuum-dried with the rotary evaporator to achieve a concentrated mixture.

Preparation of Samples.
Te beef was thawed and cut into 50 g pieces under refrigeration conditions (3 to 4 °C).Tese were subjected to 5 diferent treatments: 3% NaCl, 2% M. oleifera, 4% M. oleifera, 8% M. oleifera, and control.Tese were sealed in airtight Ziploc bags and stored at a temperature of 4 °C for 10 days.Microbiological analysis was performed on the samples in duplicates every 48 hours.

Microbiological Analysis.
Te analyses of mesophilic and coliform counts, as well as the identifcation and enumeration of Staphylococcus aureus, Escherichia coli, and Salmonella sp., were conducted using the procedures outlined by the Nordic Committee on Food Analysis (NMKL).Ten (10) grams of each beef sample were serially diluted (10 −1 to 10 −6 ) in tenfolds using sterile saline water.A homogenized suspension of each sample was prepared in a stomacher bag by mixing for two minutes and an aliquot (1 ml) of each suspension was plated.Further analyses were performed as follows.

Mesophilic Count Determination.
Te NMKL No 86 protocol was employed in the determination of the mesophilic count.Sterile Petri dishes were flled with 1 ml aliquots of each series of dilutions and molten Plate Count Agar (PCA).After gently swirling to form even mixtures, each was allowed to solidify.Tese were incubated at 30 °C for 48 hours and the formed colonies were counted [24].
2.6.Coliform Determination.Te NMKL No 44 protocol was employed in the determination of the coliform count.A 1 ml aliquot of each dilution was transferred into a sterile Petri dish and molten Violet Red Bile Agar was added.Each mixture was swirled to form a homogenous suspension and allowed to solidify.Tese were incubated for 24 hours at 37 °C and 44.5 °C.Enumeration was performed for the red colonies that were formed.In Brilliant Green Bile Broth and Escherichia coli Broth, the presence of the colonies was confrmed by gas production [24,25].

Staphylococcus aureus Determination.
Te NMKL No 66 protocol was employed in the enumeration of Staphylococcus aureus.Petri dishes containing molten Baird-ParkerAgar were flled with aliquots of each dilution and swirled to obtain even mixtures.Tese were incubated at a temperature of 37 °C for 24 hours.Staphylococcus aureus were counted and confrmed by coagulase-positive test [26].Journal of Food Quality the enumeration was carried out by using the most probable number (MPN).

Determination of
2.9.Salmonella spp.Detection.Te NMKL No 71 protocol was employed in the identifcation and enumeration ofSalmonella spp.by homogenizing 25 g of each beef sample in a stomacher bag with 225 ml of saline water and incubating each for 24 hours at a temperature of 37 °C.Aliquots of 1 ml were transferred into 10 ml of tetrathionate broth base and incubated at 37 °C for 24 hours.Also, 0.1 ml of each inoculum was transferred into Rappaport-Vassiliadis Soya Peptone Broth (10 ml) and incubated for 24 hours at 41.5 °C.
Colonies from both media were plated on Bismuth Sulfte Agar and incubated at 37 °C.Tese were examined after 24 hours [28].

Sensory Analysis.
Te treated beef samples were boiled for 25 minutes and allowed to cool at room temperature during each storage day (2, 4, 6, 8, and 10 days).Te 5-point hedonic scale (1 � dislike very much, 2 � dislike slightly, 3 � neither like nor dislike, 4 � like slightly, and 5 � like very much) was used to determine the organoleptic properties of the samples: thirty (30) untrained panel were used for the evaluation of the meat samples in terms of color, aroma, texture, taste, and overall acceptability.After tasting each meat sample, the panelists were given portable drinking water for mouth rinsing.

Statistical Analysis.
Te Minitab statistical software version 21 was used for the two-way ANOVA and comparisons among treatments and interactions with the storage.Te mean data ± SD (standard deviation) was computed.At p < 0.05, diferences between means were statistically signifcant.

Results
All the beef samples had mesophilic counts ranging from 2.0 × 10 3 CFU/ml to 8.9 × 10 7 CFU/ml from 2 to 10 days of storage (Table 1).Analysis of variance showed a signifcant diference within the beef treated with 3% NaCl.Multiple comparison tests with beef seasoned with MELE and 3% NaCl showed no signifcant diference (p > 0.05) from the 2 nd to 8 th day of storage.However, when compared to the control, there was a signifcant diference (p ≤ 0.05) from the 2 nd to the 10 th day of storage.Te mesophilic counts increased with decreased concentration of Moringa leaf extract, while it increased during storage from day 2. Also, the mesophilic count was decreased in the MELE-seasoned beef when compared to the 3% NaCl-cured beef.Te control recorded the highest microbial load during storage from day 2. Te total coliform growth (Table 2) was signifcantly higher (p < 0.05) in the control than in the treated samples.Multiple comparison tests showed that the total plate counts for beef seasoned with MELE, and 3% NaCl were not signifcantly diferent (p > 0.05) during storage from the second to the fourth day.For all treatments, the coliform counts decreased from day 0 to day 2.However, for all the treatments there was an increase in coliforms from day 2 to day 10.Also there was a decrease in coliform on days 8 and 10 for 3% NaCl and 2% MELE.Te coliform count was lower in the MELE beef treatments than in the beef cured with 3% NaCl from day 2 to 10.
Staphylococcus aureus grew on all the beef samples (Table 3).Te control recorded higher levels ranging from 2.0 × 10 4 ± 0.2 to 7.0 × 10 5 ± 0.3 from day 2 to 10. Growth was observed across all the diferent incubation periods, but a dosedependent antibacterial activity of the M. oleifera-treated beef was observed for S. aureus with the maximum inhibition recorded for 8% MELE-beef treated samples.
Tere was no growth of Escherichia coli in all the MELEtreated beef samples throughout the storage period (Table 4).
Information on the products' taste, aroma, appearance, texture, and overall acceptability is exhibited in Table 6.Te sensory parameters were graded on a scale of 1 to 5.

3.1.
Taste.Te investigation on taste revealed a clear distinction between the various beef treatments.Tere were signifcant diferences (p > 0.05) for samples treated with 8% MELE extract.However, there was no discernible variation in taste between the samples on day 0 and day 2 (p > 0.05).Similarly, there were no signifcant changes for samples on days 4, 6, 8, and 10.At 4% treatment levels, there were signifcant variations in the storage days (p > 0.05).However, there were no variations between the samples collected on days 0 and 2. While samples on days 0, 2, 4, and 6 did not reveal any signifcant diferences (p > 0.05), treatments at 2% did indicate signifcant diferences (p > 0.05).Tere were no substantial alterations for samples held for 0, 4, 6, or 10 days and for samples treated with 3% NaCl.
Te study, however, indicated that there were no differences between control samples and samples treated with 8% MELE and stored for 6 days, 8 days, and 10 days, respectively.Results also showed that the taste of the control samples at days 0, 2, and 4 was liked by the sensory panel.However, the taste of the samples stored for 4, 6, 8, and 10 days as well as control samples for 6, 8, and 10 days were not liked by the panel.

Aroma.
Tere were no diferences between the days of storage and samples treated with 8% MELE, 4% MELE, and 3% NaCl (p > 0.05).However, the samples treated at 2% MELE and the storage days showed signifcant variations (p > 0.05).Moreover, storage days and the control samples showed statistically signifcant diferences (p > 0.05).Te study's fndings revealed that samples treated at 2% MELE had the highest ratings for aroma.Te panel did not like the samples' aroma after the 8% MELE treatment.Te control samples at days 0 and 2 were, however, liked by the panel.

Appearance. Te analysis of variance in appearance
revealed that the sample treatment and storage days had a signifcant impact (p > 0.05).Te study's fndings also Journal of Food Quality 5.0 × 10 3 ± 0.0 B 5.0 × 10 3 ± 0.0 B 5.0 × 10 3 ± 0.0 B 5.0 × 10 3 ± 0.0 B 5.0      demonstrated that the panel did not like the way the samples treated at 8% MELE looked.On the other hand, the panel preferred the control samples on days 0 and 2. Te results demonstrated that the panel appreciated samples that were given 2% MELE treatment on days 0, 2, 4, and 10.Te sensory panel also preferred samples that had received 3% NaCl treatments on days 2 and 4.

3.4.
Texture.Te texture of the samples varied signifcantly (p > 0.05) depending on the diferent treatments and storage days.Te research revealed that samples treated with 8% and 4% MELE were preferred by the panel.Nonetheless, the panel preferred the samples that had been exposed to 2% MELE on days 0, 2, and 4. Except for samples on day 10, all samples that were treated with 3% NaCl were well-liked by the panel.Te panel favored the texture of the control sample on day 0 but disliked the control samples' texture on days 8 and 10.
3.5.Overall Acceptability.Signifcant variations (p > 0.05) between the sample treatments and the storage days were present.Te panel did not like any of the products treated with 8% and 4% Moringa.Te panel preferred samples that received 2% MELE treatment on days 0 and 4. Te panel also preferred the control sample on days 0, 2, and 4. On days 6, 8, and 10, the panel did not like the control samples.

Discussion
Tis work focused on the microbiological and organoleptic quality of beef seasoned with Moringa oleifera leaves extract during storage and has shown that MELE exhibits antibacterial activity which can be exploited in food systems without adversely afecting the sensory attributes if used in relatively lower concentrations.Higher concentrations of MELE can be used to achieve a greater activity against foodspoilage bacteria without compromising safety since M. oleifera has been proven to have low toxicity.Intake has been proven to be safe at supra-supplementation levels ≤1,000 mg/kg, which is far below the quantity tested in this study, when used as a nutraceutical [29].Generally, although the microbial load increased over the storage period from the 2 nd day of storage, treated beef samples had relatively lower microbial loads as well as lower coliform counts than the control samples.Also, MELEtreated samples had relatively lower microbial loads than samples treated with 3% NaCl, especially at higher concentrations.Te higher the concentration of the MELE, the slower the multiplication of the microorganisms in the samples during the storage and analysis period.Te efect of MELE on the microbial load can be attributed to the fact that Moringa oleifera leaves are rich in various phytochemical compounds including glucosinolates [30] and polyphenols such as favonoids, tannins, and phenolic acids [31].Polyphenols are known to demonstrate broad-spectrum 4.9 ± 0.4 A 4.9 ± 0.6 AB 5.0 ± 0.1 A 5.0 ± 0.1 A 5.0 ± 0. antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi [32].Although the mechanism of action of polyphenols against bacteria has not been fully explained, the antibacterial activity has been attributed to a disruption of the bacterial cell membrane [33].Similar, results have been reported by Mhalla et al. [11] which showed that the addition of extract from Rumex tingitanus to raw bovine minced meat retarded the growth of mesophilic and psychrophilic bacteria.In addition, the same study showed that the shelf life of pork sausage was enhanced when treated with the extract obtained from Citrus sp.[34].
Higher concentrations of MELE kept the growth of Staphylococcus aureus in beef at relatively lower microbial loads over the storage and analysis period as was observed in the total plate counts.Te reduced Staphylococcus aureus load after treatment of the meat with MELE suggested that the MELE can be used as a potent organic plant extract for the preservation of meat and meat products against this bacteria.However, MELE appeared to be less efective in reducing the growth of Staphylococcus aureus when compared to its efcacy against Escherichia coli.Similarly, Bouarab-Chibane et al. [33] have shown that polyphenols exhibit a species-dependent antibacterial activity on Gramnegative and Gram-positive bacteria.Te diferences in susceptibility of Staphylococcus aureus and Escherichia coli to the MELE may be attributed to the diferences in the hydrophobicity of their cell wall [27].Te surface charge of Gram-negative bacteria has been reported to be less negative in the presence of the two acids, while that of Gram-positive bacteria remains unchanged [28].Although this may be the case, studies have shown that plant extracts can efectively abrogate the growth of not only food-borne microorganisms such as Listeria monocytogenes, Pseudomonas sp., and fungi but also Staphylococcus aureus [21][22][23].Tis implies that MELE concentration above 8% may be required to signifcantly reduce the growth of some foodborne pathogens such as Staphylococcus aureus on beef during storage.
MELE efectively eliminated the growth of Escherichia coli in the beef samples during the storage and analysis period.Te current observation is in concordance with the study by Zhao et al. [35] in which the addition of Perilla frutescens leaf extract signifcantly reduced the growth of Escherichia coli in surimi fsh balls during storage at 4 °C.Roila et al. [26] also reported a similar fnding in which the incorporation of olive mill wastewater into cheese retarded the growth of Enterobacteriaceae and resulted in a significant extension of the shelf life.Te observed progressive increase in the population of Escherichia coli over the 10day incubation period for both the control and the NaCltreated meat samples suggests that MELE may improve the shelf life of meat samples more than NaCl, especially in situations where Escherichia coli has been identifed as the major contaminant during production.Food-derived polyphenols are natural preservatives and are less likely to cause the side efects associated with synthetic preservatives [20].It may also serve as a replacement for preserving meat for those on salt-free diets.Apart from this, MELE may also have a possible application in the treatment of meat before cold storage as it may potentially contribute to a reduction in the activity of psychrophilic and psychrotrophic bacteria during preservation as well as in the process of thawing before use.
Te signifcant impact of beef seasoned with MELE on the favor, aroma, appearance, texture, and general acceptability when compared to the control and 3% NaCl confrms fndings by Rahman et al. [20] which indicated that goat meat nuggets treated with 0.3% MELE during frozen storage had signifcantly (p > 0.05) improved color, favor, softness, juiciness, and overall acceptability compared to the control and other goat meat nuggets treated with 0.1% butylated hydroxyanisole (BHA).Te 8% MELE-seasoned beef was the least well-liked beef product while the 2% MELE was highly favored.Tis corresponds to investigations carried out by Evivie et al. [17] which revealed that soy meatballs treated with higher concentrations of M. oleifera leaves powder were not accepted by the panelist.However, the meatballs with lower concentrations of M. oleifera powder were accepted.Additionally, the addition of M. oleifera leaf powder to chicken patties up to concentrations of 50 g/kg did not afect the overall acceptability and other sensory parameters of chicken patties.However, concentrations above the 50 g/kg had the opposite efect [18].
Te fndings of this study concur with those of Abdallah et al. [16].In their study, samples of beef that had been treated with 2% Moringa sp. received top marks for scent and favor.M. oleifera leaves are abundant in polyphenols, carotenoids, favonoids, and other bioactive compounds that enhance food favor and aroma [36].Nevertheless, large amounts of these components are unpleasant, as found in pito samples by Ayirezang et al. [15].Although increased concentrations of MELE will have a favorable efect on reducing and eliminating food-borne microorganisms, sensory-wise lower concentrations are preferred.

Conclusion
MELE exhibits antibacterial activity which can be exploited in food preservation without adversely afecting the sensory attributes if used in relatively lower concentrations.Also, it has the potential to be used as a food preservative against meat spoilage associated with Escherichia coli but may be less efective against Staphylococcus aureus as a meat preservative.Although higher concentrations of MELE may be more efective in slowing down microbial growth in meat, these also reduce the consumer preference, and this may be a possible limitation to its application in the preservation of meat.
Te synergistic combination of lower concentrations of MELE and 3% NaCl should be studied as a way of enhancing the stability of meat without adversely afecting the sensory attributes for possible application in food systems.
Escherichia coli.Te NML No 125 protocol was employed in the enumeration of Escherichia coli.Samples from the positive Escherichia coli Broth tubes were transferred into tryptone water and incubated at 44 °C for 48 hours.Indole tests were performed by adding 3 drops of Kovac's reagent to each test culture.Te formation of a red color indicated the presence of Escherichia coli [27] and 2

Table 2 :
Mean CFU/ml of the total coliform count of raw beef seasoned with Moringa oleifera leaves extract (MELE) during storage.

Table 3 :
Mean (CFU/ml) of Staphylococcus aureus on raw beef seasoned with MELE.

Table 4 :
Mean (CFU/ml) of Escherichia coli on raw beef seasoned with MELE.
Values are means ± standard deviation of duplicate determinations.ND, not detected.

Table 5 :
Mean (CFU/ml) of Salmonella sp. on raw beef seasoned with MELE.

Table 1 :
Mean CFU/ml of total plate counts of raw beef seasoned with Moringa oleifera leaves extract (MELE) during storage.

Table 6 :
Organoleptic properties of cooked beef seasoned with MELE.
Values are expressed as means.Values within interactions of individual sensory attributes that do not share a letter are signifcantly diferent (p < 0.05).