Microbial dynamics, risk assessment and sensory evaluation of metata ayib (traditional spiced fermented cottage cheese)

Description of the study area

This study was conducted at the University of Gondar, Northwestern Ethiopia. The city is situated 723 kilometers from Addis Ababa, the capital of Ethiopia, and 32 kilometers north of Lake Tana in the Amhara Region. Gondar is situated at a height of around 2215 meters above sea level, 12° 36′ 0″ latitude, and 37° 28′ 0″ longitude. It receives between 800 and 1000 mm of rainfall annually on average (Geremew et al., 2015).

Design of the study

The cross-sectional laboratory-based experiment was carried out from January to August 2022 to detect microbial dynamics, organoleptic properties and growth potential of selected foodborne pathogens in metata ayib.

Collection of samples

Traditionally prepared ayib (3 kg) within an ice-box (at 4^oC) and various spices were transported to the microbiology laboratory of the University of Gondar. Samples were kept in a refrigerator until the preparation of metata ayib.

Preparation of metata ayib in laboratory

Metata ayib types were prepared in the laboratory based on Andualem et al. (2013); Seifu (2013) and information acquired from producers in west Gojjam (Dangila) and Gondar; 2–9 g NaCl, 2 g each of finely ground black mustard seeds (Brassica nigra) or Ethiopian mustard seed (Brassica carinata) and coriander (Coriandrum sativum) were mixed with 500 g ayib after which the whey was drained for three more days. Then the curd was mixed with 11 additional common spices (2 g each): zingebile (Zingiber officinale), korerima (Aframomum corrorima), garlic (Allium sativum), basil (Ocimum basilicum), thyme (Thymus serrulatus), rue (Ruta graveolens), bishop’s weed (Trachyspermum ammi), black cumin (Nigella sativa), shallot (Allium cepa), fenugreek (Trigonella foenum-graecum) and turmeric (Curcuma longa) or red pepper (Capsicum annuum). Some producers said that instead of Brassica nigra they were using Brassica carinata. In the preparation of Gondar metata ayib, red pepper (Capsicum annuum) is used instead of turmeric (Curcuma longa). Therefore, three different types of metata ayib (based on utilization of Brassica nigra or Brassica carinata, or Capsicum annuum separately and an additional 12 common spices) were prepared in the laboratory with a fermentation period of 20 days. Samples for microbiological analysis were taken periodically, and on the 20^th day sensory evaluation were carried out. Finally, pathogenic bacteria growth potential tests were performed on the preferred metata ayib among the three types.

Microbiological analysis of metata ayib along with fermentation period

Microbiological analysis was carried out according to Andualem et al. (2013); metata ayib samples were taken every four days to assess the microbial profile or dynamics during the fermentation process starting from day 0. During the fermentation period of metata ayib, a 10-g sample was homogenized for two min in 90 ml sterilized peptone water (0.1%, w/v). Serial dilutions (10^-1–10^-10) were prepared from this homogenized sample under a horizontal laminar flow cabinet with 0.1% (w/v) peptone water and the dilution was plated in duplicate on the specific media required for the various microbial groups to be examined. The colony forming units per gram (CFU/g) of the original samples were determined by multiplying the counts obtained with the dilution factor (Roberts and Greenwood, 2003; Goldman and Green, 2009).

Aerobic mesophilic bacteria count: An appropriate amount (0.1 ml) of serially diluted sample at the appropriate dilution level was spread on pre-solidified sterilized plate count agar (PCA) and evenly distributed with a sterilized bent glass rod. The plated samples were inverted and incubated at 30°C for 48 h. Colonies were counted by using a colony counter (Roberts and Greenwood, 2003; Goldman and Green, 2009).

Total coliform count: A serially diluted sample (0.1 ml) at an appropriate dilution level was evenly spread on properly boiled pre-solidified violet red bile agar (VRBA) with a sterile bent glass rod. The inoculated plates were inverted and incubated at 32°C for 24 h. Finally, colonies were counted by using a colony counter. Typical dark red colonies were considered as coliform (Roberts and Greenwood, 2003; Goldman and Green, 2009).

Lactic acid bacteria count: A serially diluted sample (0.1ml) at an appropriate dilution level was evenly spread on sterilized and pre-solidified de Man, Rogosa and Sharpe (MRS) agar with a sterile bent glass rod. The inoculated plates were inverted and incubated at 37°C for 72 h within an anaerobic jar. Finally, colonies were counted by using a colony counter (Roberts and Greenwood, 2003; Goldman and Green, 2009).

Staphylococcus aureus count: A serially diluted sample (0.1 ml) at an appropriate dilution level was evenly spread on sterilized and pre-solidified mannitol salt agar (MSA) with a sterile bent glass rod. The inoculated plates were inverted and incubated at 37°C for 48 h. After incubation, plates with 30 to 150 colonies was counted (Roberts and Greenwood, 2003; Goldman and Green, 2009).

Mold and yeast count: A serially diluted sample (0.1 ml) at an appropriate dilution level was evenly spread on sterilized and pre-solidified potato dextrose agar (PDA) supplemented with chloramphenicol. Inoculated plates were inverted and incubated at 25°C for five days. After incubation, colonies were counted with a colony counter (McLandsborough, 2005).

Sensory analysis

Sensory evaluation of metata ayib varieties based on the difference in utilization of spices (Brassica nigra, Brassica carinata or Capsicum annuum, all in addition to 12 other common spices) was conducted according to the method described by Resurrecin (1998). Sensory attributes considered included color, aroma, taste and overall acceptability. A total of 10 adult metata ayib consumers were requested to evaluate the sensory attributes of the metata ayib samples and they took part voluntarily; and participants gave informed oral consent for their data to be used. The following criteria were used to choose the members of the consumer panel: they must be “consumers” of metata ayib and be between the ages of 18 and 40. Samples of metata ayib (20 g) were served in a well-lit, ventilated space on white plastic plates with a three-digit code. Water in bottles was provided to the panelists to rinse their mouth after each sample. The taste, color, aroma, texture, appearance and overall acceptability of metata ayib samples were evaluated using a 5-point Hedonic scale (5 = like very much, 4 = like moderately, 3 = neither like nor dislike, 2 = dislike moderately and 1 = dislike very much). The metata ayib samples were presented in a random fashion.

Pathogenic bacteria growth challenge test in metata ayib

The in vitro pathogen inhibition study was performed in triplicate according to Millette et al. (2007) and Nemo and Bacha (2021), with some modifications. Each of the standard pathogens, E. coli ATCC 25922, S. aureus ATCC 25923, and C. albicans ATCC 10231,was cultivated at 35–37^oC for 24–48 h in Mueller Hinton broth. Accordingly, 100 g metata ayib prepared in a laboratory was treated at 80°C for 10 min. to kill any vegetative cells that might be present in the metata ayib. Then, 1 ml of an overnight culture of the selected pathogenic microbe was inoculated onto pasteurized metata ayib. The content of the pathogen was estimated by spectrophotometric measurements at 600 nm. Finally, the growth potential of each pathogenic microbe was monitored on a regular basis by homogenizing 10 g metata ayib in 90 ml buffered peptone water, spreading 0.1 ml of the appropriate dilution on plates, and counting the colonies on eosin methylene blue (EMB) agar for E. coli, MSA for S. aureus, and PDA supplemented with chloramphenicol for C. albicans. Metata ayib samples were taken at 0 h, 6 h, 12 h, 18 h, and 24 h in order to evaluate the concentration of each pathogenic microbe during fermentation.

Data analysis

Prior to statistical analysis, microbial count data were first converted to logarithmic values (log10). The one-way ANOVA procedure using SPSS software (RRID:SCR_002865) was used to analyze the transformed microbial count data along the fermentation period of each of the three types of metata ayib with four-day intervals, the preference based on sensory evaluation, and the growth potential of some food-borne pathogens in metata ayib, separately. The values were considered to be significantly different at 95% (P<0.05). Using the least significant difference, the mean separation was calculated.

Microbial dynamics of main microbial groups of metata ayib (prepared with black mustard seed and 12 additional spices) sample during fermentation period

The sample taken on day 0 (8.989±0.011 CFU/g) for the determination of aerobic mesophilic bacteria count (AMBC) (Table 1) was significantly different (P<0.05) from the samples taken on the 4^th (7.945±0.779 CFU/g) and 12^th days (7.037±0.015 CFU/g), and it was decreasing. When the results of the 16^th (8.709±0.390 CFU/g) and 20^th (8.755±0.03 CFU/g) days were compared with the day 0 result (8.989±0.011 CFU/g), the AMBC was decreasing but not significantly (P>0.05).

Metata ayib samples made with black mustard seed and 12 additional common spices were used to determine lactic acid bacteria count (LABC) (Table 1), and a sample at day 0 (8.915±0.013 CFU/g) was significantly different (P<0.05) from samples at the 4^th (8.238±0.267 CFU/g), 8^th (7.935±0.252 CFU/g), 12^th (7.81±0.176 CFU/g), 16^th (7.518±0.340 CFU/g) and 20^th (7.532±0.128 CFU/g) day of fermentation and it was decreasing. When compared with the 16^th day of fermentation, the sample taken on the 20^th sampling day revealed a modest increase in LABC, although there was no statistically significant difference (P>0.05).

The yeast and mold count (YMC) was calculated from a metata ayib sample made with black mustard seed and 12 additional common spices (Table 1), and a sample taken at day 0 (7.579±0.03 CFU/g) was significantly different from the 4^th (6.282±0.613 CFU/g), 12^th (4.713±0.029 CFU/g), 16^th (5.529±0.051 CFU/g) and 20^th (6.778±0.029 CFU/g) fermentation days. The YMC dropped over the course of the fermentation process, but from the 12^th day of sampling to the 20^th day, it considerably increased (P<0.05).

Microbial dynamics of main microbial groups of metata ayib (Ethiopian mustard seed and 12 additional common spices) sample during fermentation period

AMB were detected from metata ayib made with Ethiopian mustard seed and 12 additional common spices (Table 2), and the first (9.005±0.013 CFU/g) and fourth (8.733±1.187 CFU/g) sampling days were significantly different from the sample taken at the 16^th (6.832±0.026 CFU/g) sampling day and it was decreasing. When the day 0 (9.005±0.013 CFU/g) sample was compared with the 20^th sampling day (8.237±0.933 CFU/g), the result was decreasing but not significantly (P>0.05). However, the AMBC rose from the 16^th (6.832±0.026 CFU/g) to the 20^th (8.237±0.933 CFU/g) sampling day with a significant difference (P<0.05).

With the LABC determined from the sample of metata ayib that was prepared with Ethiopian mustard seed and 12 additional common spices (Table 2), the day 0 fermentation sample (8.913±0.032 CFU/g) was significantly different (P<0.05) from the 8^th (7.574±0.826 CFU/g), 16^th (7.913±0.021 CFU/g) and 20^th (7.371±0.374 CFU/g) fermentation days. The LABC on the first day (8.913±0.032 CFU/g) was larger than the 20^th day (7.371±0.374 CFU/g), and the difference was statistically significant (P<0.05).

The YMC was calculated for the sample of metata ayib which was made from Ethiopian mustard seed and 12 additional common spices (Table 2). The sample’s load on the first day of fermentation (7.59±0.033 CFU/g) was significantly different (P<0.05) from samples taken on the 4^th, 16^th, and 20^th days of fermentation (6.249±1.171 CFU/g, 5.747±0.031 CFU/g, and 6.578±0.734 CFU/g, respectively), and it was decreasing.

Microbial dynamics of main microbial groups of metata ayib (red pepper and 12 additional common spices) sample during fermentation period

For the metata ayib sample made from red pepper and an additional 12 common spices (Table 3), the AMBC determined on day 0 of fermentation (8.999±0.013 CFU/g) was significantly different (P<0.05) from the AMBC identified on the final (20^th) sampling fermentation day (6.361±0.423 CFU/g), and it was lower.

The day 0 fermentation sample of the metata ayib made with red pepper and 12 additional common spices (Table 3) was significantly different (P<0.05) from the samples taken on the 4^th, 8^th, 12^th, 16^th and 20^th fermentation days (7.316±0.299 CFU/g, 6.972±0.246 CFU/g, 8.149±0.012 CFU/g, 7.954±0.019 CFU/g and 6.733±0.51 CFU/g, respectively), and it decreased.

The metata ayib sample prepared with red pepper and 12 additional common spices (Table 3) was used for YMC determination, and the sample taken on the first fermentation day (0) (7.602±0.022 CFU/g) was significantly different from samples taken on the 4^th, 8^th, 12^th, 16^th, and 20^th fermentation days (6.700±0.774 CFU/g, 6.591±0.011 CFU/g, 6.845±0.012 CFU/g, 5.959±0.01 CFU/g and 5.982±0.009 CFU/g, respectively). In comparison with the first fermentation day (7.602±0.022 CFU/g), the 20^th fermentation day (5.982±0.009 CFU/g) showed a significant decrease (P<0.05).

Metata ayib is made from fermented ayib and various spices, which may have an inhibitory effect due to the antimicrobial activity of the spices, altered physico-chemical activity, and metabolites released by various microorganisms. Coliforms and Staphylococci species were not detected in any of the metata ayib types. This finding is consistent with studies on metata ayib conducted by Andualem et al. (2013) and Seifu (2013), as well as those on tej conducted by Bahiru et al.(2006). However, Nemo and Bacha (2021) reported that growth of coliforms and Staphylococci species were detected throughout the fermentation period of Ethiopian traditional fermented beverages (Borde, Grawa, and tej), though they decreased in all beverages during fermentation. Coliforms were likewise reduced to undetectable levels during ergo (Gonfa et al., 1999) and maize-based shameta fermentation (Kitessa et al., 2022).

The results of all microorganism groups counted on the 20^th sampling day demonstrated that they were reduced compared with the first sample day (Tables 1, 2, 3). In comparison with metata ayib prepared with red pepper, Ethiopian mustard seed, both with 12 additional common spices, metata ayib prepared with black mustard seed and 12 additional common spices revealed the largest load of microbe groups (AMBC, LABC, and YMC) at day 20 of fermentation. Given that the amounts of LABC and other groups of bacteria were relatively high on the final sampling day (20^th), it is possible to claim that metata ayib made with black mustard seed and 12 additional common spices may be utilized as a probiotic food. The decrease in microbial groups may be caused by alteration in physico-chemical characteristics, restriction of nutrients, and development of antimicrobial agents from the beginning to the end of the fermentation process; this is consistent with research on a variety of fermented foods from Ethiopia, including Idris et al. (2001) on awaze and datta; Andualem et al. (2013) on metata ayib; Berhanu (2014) on tella; Karssa et al. (2014) on kocho; Nemo and Bacha (2021) on three Ethiopian alcoholic beverages; and Kitessa et al. (2022) on shameta.

The AMBC of metata ayib prepared with black mustard seed, Ethiopian mustard seed and red pepper, all with 12 additional common spices, were 8.989±0.011 to 8.755±0.03 CFU/g (P>0.05), 9.005±0.013 to 8.237±0.933 CFU/g (P>0.05) and 8.999±0.013 to 6.361±0.423 CFU/g (P<0.05), respectively. When compared with metata ayib prepared with red pepper and 12 additional common spices (6.361±0.423 CFU/g) and Ethiopian mustard seed and 12 additional common spices (8.237±0.933 CFU/g), preparations made with black mustard seed and 12 additional common spices had the highest AMBC load at the 20^th sampling day (8.755±0.03 CFU/g). These results are slightly higher than those of Seifu (2013), who reported total bacterial counts ranging from 2.5 × 10⁵ to 6.9 × 10⁷ CFU/g from metata ayib, which may be due to differences in the quality of cottage cheese and variation of spices. They are also higher than the results of Bahiru et al. (2006), who reported an AMBC <3 log CFU/ml in the tej sample, and Bacha et al. (2010) who recorded 5.69±0.79 CFU/g, in a wakalim sample.

The LABC of metata ayib prepared with black mustard seed, Ethiopian mustard seed and red pepper, all with 12 additional common spices, were 8.915±0.013 to 7.532±0.128 CFU/g (P<0.05), 8.913±0.032 to 7.371±0.374 CFU/g (P<0.05) and 8.912±0.042 to 6.733±0.51 CFU/g (P<0.05), respectively. At the 20^th sampling day, metata ayib prepared with black mustard seed and 12 additional common spices had the highest load of LABC (7.532±0.128 CFU/g), compared with metata ayib prepared with Ethiopian mustard seed and 12 additional common spices (7.371±0.374 CFU/g) and red pepper and 12 additional common spices (6.733±0.51 CFU/g). These results are higher than those of Bacha et al. (2010), who recorded 4.42±0.05 CFU/g in wakalim; Bahiru et al. (2006), who reported mean counts > 6 log CFU/ml in tej; and Kitessa et al. (2022), who reported 8.33 log CFU/g in shameta. This result is somewhat consistent with Nemo and Bacha’s (2021) report of a LAB load of 7.33.

The YMC of metata ayib prepared with black mustard seed, Ethiopian mustard seed, red pepper, all with 12 additional common spices, were 7.579±0.034 to 6.778±0.029 CFU/g (P<0.05), 7.59±0.033 to 6.578±0.734 CFU/g (P<0.05) and 7.602±0.022 to 5.982±0.009 CFU/g (P<0.05), respectively. These results are comparable with those of Bahiru et al. (2006) and Kitessa et al. (2022), who reported yeast counts >6 and 6.44 log CFU/ml, respectively. However, they are higher than those reported by Bacha et al. (2010), who reported 4.08±0.34 CFU/g in wakalim, and Seifu (2013), who reported results ranging from 1.2 × 10² to 6.1 × 10³ CFU/g in metata ayib. The variation in results may also be because of the variety of spice types and variations in the quality of cottage cheese used in the fermentation process.

On the 20^th sampling day, metata ayib prepared with black mustard seed and 12 additional common spices had the greatest load of YMC (6.778±0.029 CFU/g), followed by metata ayib prepared with Ethiopian mustard seed and 12 additional common spices (6.578±0.734 CFU/g) and red pepper and 12 additional common spices (5.982±0.009 CFU/g).

Paired t-test results on the 20^th fermentation day

Paired t-test results for AMBC (Table 4) of metata ayib (at 20^th fermentation day) samples prepared with black mustard seed and Ethiopian mustard seed, both with 12 additional common spices, showed a positive correlation coefficient (0.723), while metata ayib prepared with black mustard seed and red pepper, both with 12 additional common spices, showed a negative correlation coefficient (−0.303), and metata ayib prepared with Ethiopian mustard seed and red pepper, both with 12 additional common spices, showed a negative correlation coefficient (−0.878) but there was no significant difference (P>0.05) of AMBC among all types of metata ayib.

Paired t-test results for LABC (Table 4) of metata ayib (at 20^th fermentation day) samples prepared with black mustard seed and Ethiopian mustard seed, both with 12 additional common spices, showed a negative correlation coefficient (−0.956) and were not significantly different (P>0.05), and metata ayib samples prepared with black mustard seed and red pepper, both with 12 additional common spices, showed a negative correlation coefficient (−0.939), and were not significantly different (P>0.05), while metata ayib (at 20^th fermentation day) samples prepared with Ethiopian mustard seed and red pepper, both with 12 additional common spices, showed a positive correlation (0.999) and was significantly different (P<0.05).

Paired t-test results for YMC of metata ayib (at 20^th fermentation day) samples (Table 4) prepared with black mustard seed and Ethiopian mustard seed, both with 12 additional common spices, showed a positive correlation coefficient (1.000) and were significantly different (P<0.05), while metata ayib samples prepared with black mustard seed and red pepper, both with 12 additional common spices, showed a negative correlation coefficient (−0.511) and were not significantly different (P>0.05), and metata ayib samples prepared with Ethiopian mustard seed and red pepper, both with 12 additional common spices, showed a negative correlation coefficient (−0.521) and were not significantly different (P>0.05).

Sensory evaluation of traditional metata ayib varieties

The sensory evaluation results of 10 consumer panelists are presented in Table 5. The results reveal that the metata ayib prepared with black mustard seed and 12 additional common spices tasted considerably (P<0.05) better than that prepared with red pepper and Ethiopian mustard seed, both with 12 additional common spices.

Metata ayib prepared with black mustard seed and 12 additional common spices did not significantly differ in terms of color, aroma, texture, appearance, or overall acceptance (P>0.05), but it scored slightly higher than metata ayib prepared with Ethiopian mustard seed, and 12 additional common spices in all categories. Metata ayib prepared with black mustard seed and 12 additional common spices differed significantly from metata ayib prepared with red pepper and 12 additional common spices in all organoleptic qualities. The results of metata ayib prepared from Ethiopian mustard seed and red pepper, both with 12 other common spices, did not differ significantly (P>0.05) in terms of color or texture, but they did (P<0.05) in terms of aroma, appearance, and acceptance.

The organoleptic properties result of metata ayib prepared with black mustard seed, and 12 additional common spices was slightly higher than the result for metata ayib reported by Eshetu and Asresie (2019), which was 4.45±0.24 to 4.8±0.23, 4.31±0.14 to 4.56±0.14, 4.75±0.15 to 4.74±0.33, 4.83±0.17 to 4.86±0.19, 4.47±0.39 to 4.57±0.37, and 4.27±0.25 to 4.33±0.17 for taste, color, aroma, texture, appearance and over all acceptability, respectively.

Growth potential of pathogens in metata ayib within 24 h

From a metata ayib sample made with black mustard seed and 12 additional common spices that was preferred based on sensory evaluation, the growth potential of C. albicans ATCC 10231, E. coli ATCC 25922 and S. aureus ATCC 25923 was observed (Table 6). E. coli inoculated into metata ayib showed a maximum load at 6 h (5.652±0.039 CFU/gm), but its growth decreased starting at 12 h to non-detectable level at 24 h and it was statistically significant (P < 0.05). The increase in C. albicans load peaked at 12 h (7.042±0.014 CFU/g), then declined from 18 h (4.152±0.012 CFU/g) to 24 h (2.785±0.028 CFU/g), and it was statistically significant (P<0.05). S. aureus was decreased at 0 h (3.456±0.902 CFU/g) and 6 h (3.007±0.476 CFU/g), but was not present from 12 h onwards till the completion of the sampling (24 h). It may be due to the release of antimicrobial metabolites by various LAB groups, yeasts and molds, as well as changes in physico-chemical activities (such as pH and water activity) and the antibacterial activity of certain spices. This outcome is comparable to that of Yilma et al. (2016), who reported decreased Salmonella typhimurium DT104 inoculated with LAB; Anteneh et al. (2011), who also reported that the mean counts of the test pathogens reduced by 4 log units at 12 h and were completely eliminated from both ready-to-consume products at 24 h; and Nemo and Bacha (2021), who demonstrated a decreased load of pathogens inoculated into three Ethiopia traditional fermented beverages.

E. coli and S. aureus were less resistant than C. albicans. S. aureus was less able to survive than E. coli. This outcome is consistent with that of Nemo and Bacha (2021). S. aureus was more sensitive than the other pathogens that were examined; this may be because Gram positive and closely related bacteria respond better to antimicrobials such bacteriocin generated by LAB than Gram negative bacteria (e.g. E. coli). To improve its safety and health benefits, fermented metata ayib should be handled with appropriate hygiene and inoculated with possible probiotics.