African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5182

Full Length Research Paper

Microbiological characterization of traditionally fermented food in southern Mozambique

Nhassengo Anisa J. de A.
  • Nhassengo Anisa J. de A.
  • Gaza Regional Veterinary Laboratory, Agricultural Research Institute of Mozambique, Mozambique.
  • Google Scholar
Massamby Andreia M. M.
  • Massamby Andreia M. M.
  • Department of Crop Production, Faculty of Agronomy and Forestry Engineering, Eduardo Mondlane University, Mozambique.
  • Google Scholar
Fulano Arsénio R.
  • Fulano Arsénio R.
  • Department of Para-Clinics, Faculty of Veterinary Medicine, Eduardo Mondlane University, Mozambique.
  • Google Scholar
Macuamule Custódia L. M.
  • Macuamule Custódia L. M.
  • Department of Para-Clinics, Faculty of Veterinary Medicine, Eduardo Mondlane University, Mozambique.
  • Google Scholar


  •  Received: 22 February 2022
  •  Accepted: 10 August 2022
  •  Published: 31 August 2022

 ABSTRACT

Traditionally fermented foods are source of income and improve quality of diets in rural communities. In Mozambique there are several locally fermented foods, however little is known about fermentation technology and microbial composition. This study aimed to determine physico-chemical parameters and the microbial diversity of traditionally fermented foods in Mozambique. Samples of ucanhi, maheu, massi and rhali were analyzed in regard to pH and titratable acidity; lactic acid bacteria (LAB), yeasts and microorganisms of food safety concern (Echerichia coli and molds). Purified isolates were identified at the species level using identification kits. The results show that in fermented foods, the pH ranged from 3.33±0.17 to 4.42±0.12 and the titratable acidity from 2.74±0.92 to 9.75±2.87. Counts ranged from 2.54±0.57 to 4.23±1.09 Log CFU/ml for LAB and 2.24±0.43 to 3.63±0.55 Log CFU/ml for yeast. Apart from ucanhi, molds were present in almost all products in quantities that reached 3.59±0.42 to 4.29±0.45 Log CFU/ml. The isolated species from the fermented products were Lactobacillus plantarum, L. fermentum, for LAB, and Candida albicans, C. famata, Cryptococcus humicola, Rhodotorula mucilaninosa 2, Saccharomyces cerevisiae, for yeasts. In general, fermented foods in Mozambique are quite acidic; LAB and yeast counts were low; microorganisms with public health importance were isolated in these products.

 

Key words: Fermented foods, food safety, lactic acid bacteria, Mozambique, yeasts.


 INTRODUCTION

Food fermentation is one of the oldest processing technologies where the growth of spoilage and pathogenic organisms are suppressed to promote the extension of the shelf life of perishable products (Terefe, 2016). Fermented foods play an important role in food security, sustainable development, and economic growth in Africa through the provision of employment facilities, contribution to empowerment initiatives for unemployed women, opportunities for scaling up of traditional food processing techniques and, distribution of resultant products (Obafemi et al., 2022).

 

Fermentation can contribute to preservation of food, actively participate in the development of their texture, flavor and aroma, help to eliminate pathogens, allergens and toxic substances, improve digestibility, create new products for new markets and increase nutrient value (Voidarou et al., 2021; Obafemi et al., 2022). The associated microbiomes of fermentations can have health-promoting properties, with some probiotic strains (Terefe, 2016; Voidarou et al., 2021).

 

Lactic acid bacteria (LAB) and yeasts belong to this group, and the predominant genus and species in foods vary according to the climatic conditions of each region (Akabanda et al., 2010). The intake of probiotics has been reported to be efficient in the prevention of several types of diarrheas and colitis in children and adults, as well as in the treatment of other gastrointestinal disorders (Syal and Vohra, 2013).

 

In Mozambique, a dual public health concern has risen in recent years. The occurrence of chronic diarrhea in both children and adults and the actual high levels of chronic malnutrition (44%) gives a picture of the current nutritional status of the national population, although, micronutrient deficiency is transversal between the urban and rural population (UNICEF, 2013). Regarding this matter, there is a need to identify natural sources of probiotics from traditional foods in southern Mozambique, which could be an aspect to consider introducing in future food fortification strategies, especially at the rural level, where high rates of chronic malnutrition have been widely reported.

 

Some authors have described the fermentation technology and microbiology of some traditionally fermented foods produced in African countries, as well as the introduction of the use of microorganisms involved in fermentation and their products in food fortification programs. Hjortmo et al. (2008), observed high levels of folate during the fermentation of togwa (maize-based fermented beverage produced in Tanzania). In amasi (food produced in South Africa and Zimbabwe, by spontaneous fermentation of milk) bacteria of the genus Lactobacillus with probiotic effect, such as L. helvetieus, L. plantarum, L. delbrueekii subsp. Lactis and Lactis e L. casei subsp. casei were isolated (Beukes et al., 2001).

 

Achi (2005) isolated Lactobacillus rhamnosus, L. reuteri and Saccharomyces cerevisiae in Ogi, a porridge produced from the fermentation of corn (Zea mays), sorghum (Sorghum bicolor) or millet (Peninselum americarum), intended for feeding babies. The same author also isolated several species of Lactobacillus and Saccharomyces cerevisiae in alcoholic beverages produced from the fermentation of sorghum called bukuruto and pito in Nigeria, and sorghum beverage in South Africa. Akabanda and collaborators (2010) isolated species of Lactobacillus (L. acidophilus and L.bulgaris), Lactococcus species (L. cremoni and L. lactis), Streptococcus thermophilus, Leuconostoc sp and Saccharomyces sp in nunu, traditionally fermented dairy beverage in Ghana. However, for the microorganisms present in fermented foods to exert their effect efficiently, their ingestion must be continuous so that they  colonize the gastro-intestinal tract (Acurcio, 2011).

 

In Mozambique there are several locally fermented foods, such as maheu (a refreshing drink made from corn flour), massi (spontaneously fermented milk), fermented fruit drinks (marula or ucanhi and cashew), among others. However, little has been described about the technology of production, microbial diversity, and the potential use of these products as natural probiotics. The present work aimed at determining the physicochemical parameters, identifying, and quantifying the micro-organisms present during the fermentation process, as well as assessing to what extent is it safe to consume traditional fermented foods, considering the microbiological quality of the final product.


 MATHERIALS AND METHODS

Description of study site

 

The study was carried out in, Maputo City (25°57'13.4064''S, 32°35'19.3596''E), Gaza (25°02'60.00"S, 33°38'59.99") and Inhambane (23°51′53″S, 35°22′59″E) provinces, located in Southern Mozambique. The provinces were selected according to their history of production of traditionally fermented foods. Twenty samples of ucanhi were acquired in Marracuene, Magude, Manhiça and Goba districts (Maputo City province), 19 samples of maheu in Chamanculo and Kamubukwana district (Maputo City province), 18 samples of massi in Chokwé and Guijá districts (Gaza province), and 20 samples of rhali in Maxixe and Inharrime districts (Inhambane province).

 

Preparation of the fermented foods

 

The ucanhi is a beverage consumed in traditional occasions in southern Mozambique. The processing of preparation starts with the manual pressing of the pulp of the marula fruit (Sclerocarya birrea) to remove the juice, followed by the placement of the liquid in plastic containers, where the juice is kept in a cool place for 2 to 3 days, to allow the fermentation to take place using the natural microflora. During the fermentation, the juice is separated into two layers, a foamy supernatant that is discarded and the liquid that is the proper ucanhi. The fermentation is the main stage and the critical point of control.

 

Maheu is a traditional beverage made by a mixture of corn flour and water in a proportion of 2:6 to obtain a porridge that is boiled for about an hour. Afterwards, the porridge is cooled at room temperature, and after cooling down the porridge is placed in a container, where sugar is added to stimulate the natural microflora. To allow the fermentation to take place, the porridge is stored at room temperature for 3 to 7 days. Maheu's processing technology has two stages that are the critical points of control: the porridge boiling and the natural fermentation.

 

The masi production is performed with the deposition of raw cow milk is plastic buckets, that are sealed and stored indoors for 2 to 3 days to allow the fermentation. The fermentation is a critical control point and is carried out relying both on the natural microflora of the milk and the container used for keeping the raw milk. During the fermentation, two layers are formed, a liquid layer (whey) that is decanted and a thick clot (massi) that is kept in the bucket for consumption.

 

The processing of rhali starts with the peeling of cassava (Manihot esculenta), followed by grating it into a fine pulp using a metal  grater.  After  that the fine pulp is placed in bags and pressed with stones or wood to remove excess of moisture, and the bags are placed at room temperature for 6 days to allow the fermentation process to occur. After 6 days the fermented pulp is mixed with cassava flour, then roasted in metal containers or clay pots heated over in open fire and after that the roasted flour is sieved according with the size of the granules. The rhali processing technology has the roasting as a main of critical control point.

 

Sampling procedure of the fermented foods and performed analysis

 

For ucanhi, maheu and massi approximately 1000ml of each product were collected per sample. For the rhali samples 1000g were collected per sample. All liquid samples (ucanhi, massi and maheu) were placed in sterile plastic bottles, properly marked, stored in coolers containing icepacks, and kept in cold conditions before transportation (-40°C). The solid samples (rhali) were placed in sterile plastic bags, marked accordingly and thereafter stored at room temperature (+25°C), before transportation. After the sampling process (approximately 4 h), all the collected samples were transported immediately to the Food Hygiene and Technology Laboratory at the Veterinary Medicine Faculty, at Eduardo Mondlane University (FAVET-UEM), for further analysis.

 

The experimental design followed a chronological order which includes physico-chemical tests (determination of pH and titratable acidity); isolation, identification, and quantification of lactic acid bacteria (LAB) and yeast; isolation, quantification and identification of E. coli and molds. All samples were analyzed in triplicate and plated in duplicate.

 

pH and titratable acidity determination

 

The pH was determined using the electrometric method (using a digital potentiometer) (Instituto Adolfo Lutz, 2008).

The determination of the titratable acidity was carried out using the volumetric titration method with an indicator (Instituto Adolfo Lutz, 2008). Based on the results obtained, the acidity was calculated based on the following formula:

 

 

where V = number of ml of 0.1 or 0.01 M sodium hydroxide solution used in the titration, f = 0.1 or 0.01 M sodium hydroxide solution factor, P = nr of grams of the sample used in the titration, c = correction factor for 1M NaOH solution, 10 for 0.1 M NaOH solution and 100 for 0.01 M NaOH solution.

 

The titratable acidity was expressed in ml of the 1N NaOH solution/100 g of sample.

 

Isolation, identification, and quantification of microorganisms

 

LAB and yeasts

 

The isolation and identification of LAB and yeasts in ucanhi, maheu, massi and rhali was carried out using the methodology described by Hellström et al. (2010) and Greppi et al. (2017). Two serial dilutions (10-1 and 10-2) were performed using peptone water, following the methodology described by Akabanda et al. (2010). From each of the dilutions obtained, an aliquot of 0.1 ml was pipetted, and surface plated on agar Petri dishes containing the culture media MRS Agar (Sigma - Aldrich) supplemented with 0. 31μg/ml Griseofulvin (Griseofulvin, from Penicillium griseofulvum, 97.0 - 102.0%; Sigma-Aldrich), to prevent yeast growth in the case of LAB; and YPD Agar (Sigma-Aldrich) supplemented with 25 mg/ml  Chloramphenicol   (Chloramphenicol  ≥ 98%  HPLC,  Sigma-Aldrich) to prevent bacterial growth in the case of yeast. All agar plates were placed inside glass jars with screw caps containing anaerobic generating sachets (Sigma - Aldrich) and inclubated at 30ºC for 48 h in the case of LAB, and at 30ºC for 72 h in the case of yeast (Hellström et al., 2010 and Greppi et al., 2017). After the incubation, the morphological characterization of the colonies (visual appearance eg. colour, size, shape, type) was performed, and the total number of the colonies per dilution was estimated using the colony count method. Dilution factor was considered, and results were expressed in CFU/g, and after that transformed to logarithm units.

 

For all plates with different morphological aspect, purification was performed by surface plating in Petri dishes containing YPD Agar (Sigma-Aldrich), supplemented with Chloramphenicol (0.25 mg/ml) [Chloramphenicol ≥ 98% (HPLC ), Sigma - Aldrich] and incubated at 30°C for 24 h for yeast; and MRS Agar (Sigma-Aldrich) supplemented with Griseofulvin (0.31 μg/ml) (Griseofulvin, from Penicillium griseofulvum, 97.0 - 102.0%; Sigma-Aldrich) and incubated in an oven in screw-top glass jars containing generator sachets of anaerobiosis (Sigma-Aldrich) at 30°C for 24 h for LAB. For LAB, the catalase test was then performed, which consisted of making smears of catalase negative colonies on slides, followed by staining by the Gram method and observation under an optical microscope using 100X resolution.

 

LAB and yeast colonies were cryopreserved following the methodology described by Nordvall (2007) and Greppi et al. (2017). All pure LAB colonies that were catalase negative and Gram positive (Gram +) were cryopreserved. Pure colonies were inoculated in cryo-tubes containing 40% glycerol (Glycerol, ≥ 99.0%, Sigma - Aldrich) in MRS broth (Sigma - Aldrich) for the case of LAB, and YPD broth (Sigma - Aldrich) for the case of yeasts, and preserved at temperature of -32°C. For the confirmatory tests, bioMérieux’s API identification products that consist of test kits for identification of Gram-positive and Gram-negative bacteria and yeast were used.

 

The API method is a quick and well-established system for manual microorganism identification to the species level. This system offers a large and robust database available online  (APIWEB™ service).

 

For the identification of yeats was used to test the API® 20 C AUX (bioMérieux’s), that is a system for the precise identification of the most frequently encountered yeasts. The API® 50 CH test (bioMérieux’s) were used as a system for the precise identification of the most frequently encountered Lactobacillus and related genera. The API identification tests were performed following the manufacturer's instructions (bioMérieux’s)

 

Microorganisms of food safety concern

 

The microorganisms of food safety concern selected for analysis were E. coli and contaminating molds. The microorganisms studied were chosen due to their better resistance to the acidic conditions of fermented foods. Detection of E. coli was performed in accordance with the International Commission on Microbiological Specifications for Foods (ICMSF, 2012). About 0.1 ml volume of the 10-1 dilution was pipetted, plated by spread on the surface of Eosin Methylene Blue Agar (EMB Agar, Sigma - Aldrich). Duplicate plates were incubated at 37°C for 24 h. For confirmation of E. coli colonies, the indol test was performed.

 

Mold isolation was carried out according to the method established by the ICMSF (2012). For this purpose, about 1 ml aliquot of the serial dilutions was taken into Petri dishes, containing about 15 ml of molten Sabouraud Dextrose Agar (SDA), followed by incubation at 30°C for 72 h. Plates were analyzed in duplicate. The colonies were estimated using a colony counter. Dilution factor was considered for the estimation of the final results, and results were expressed  in  CFU/g,  and after that transformed to logarithm units.


 RESULTS AND DISCUSSION

Physico-chemical parameters

 

Table 1 illustrates the mean values of pH and titratable acidity (expressed in ml of the 1N NaOH solution/100g of sample) in the traditionally fermented foods under study. In general, the results obtained show that traditionally fermented foods in southern Mozambique are very acidic, with 3.33 ± 0.17 to 4.42 ± 0.12, where rhali and massi stood out as more acidic foods. Titratable acidity ranged from 2.74 ± 0.92 to 9.75 ± 2.87, with the highest values reported in ucanhi and massi.

 

 

Similar results to present the study were reported by Penidoa et al. (2018) during the evaluation of the selection of starter cultures to produce cassava starch through the fermentation of cassava flour. The authors obtained pH values ranging from 3.29 to 5.69 and titratable acidity in the range of 0.14 to 0.71% in the fermented cassava flour, and the titratable acidity values referenced by them are below those observed in the present study for the case of rhali. Oyeyinka et al. (2020) during the characterization of physical, chemical, and sensory properties of flakes (gari) prepared from refrigerated cassava roots, obtained pH values ranging from 4.30 to 5.40. Gari is a fermented product with pH found to vary between 3.42 and 4.88 depending on processing methods. The acid pH contribute largely to the flavor and consequently the acceptability of rhali by consumers (Oyeyinka et al., 2020).

 

The pH ranges obtained in the present study for maheu are similar to those obtained by Mwale (2014), which reported pH values in the order of 3.5 and titratable acidity in the range of 0.4 - 0.5% for the food in reference, presenting a range that is relatively lower than that reported in the present study. Simatende (2016) during the characterization of the microflora diversity present in emahewu produced in Swaziland, obtained 3.62 as an average pH value similarly with the present study, and titratable acidity values in the order of 0.43%, relatively lower compared to the present study. Mashau et al. (2020) during the evaluation of the shelf-life extension and sensory properties of mahewu – a non-alcoholic fermented beverage, by adding Aloe vera powder, obtained pH value ranging from 2.4 to  3.3  and  titratable acidity in the order of 0.2 to 1.8%.

 

Exceptionally, among the four traditionally fermented foods under study, maheu stood out for being the least acidic food than the others (pH 3.33 ± 0.17) and for having relatively lower titratable acidity values than the other foods (2.74 ± 0.92). The low pH values obtained in some traditionally fermented foods analyzed in this study may be associated with the long fermentation time, since the cold system is not applied to stop the fermentation process. Factors such as the production of high levels of organic acids and, consequently, the accentuated sour flavor enhancement were notorious, corroborating to the results described by Nyambane et al. (2014). The low pH values obtained in the present study are crucial, since most bacteria, including the pathogenic microorganisms, struggle to grow at low pH values, and this condition provides the microbial safety, as well as the extension of the shelf life of mahewu samples.

 

The pH results obtained for massi in the present study are similar to those reported by Yu et al. (2015) also found that the mean pH of fermented cow's milk ranges from 4.12 ± 0.35 to 4.31 ± 0.39. Regarding titratable acidity, the values obtained in this study are above those mentioned by Simatende (2016), who obtained in his study a titratable acidity of 0.89% in samples of cow's milk traditionally fermented in Swaziland. The origin of these differences may be associated with the microbiological composition of the massi, the production of organic acids and the fermentation time.

 

In relation to ucanhi, the results of the study in reference are similar to those reported by Naeem et al. (2012), which reported pH mean values ranged from 2.0 to 4.5. In contrast, results reported by Motlhanka et al. (2018), showed that the pH of marula wine produced in Sub-Saharan Africa reached 4.10, a relatively higher value compared to the ones obtained in the present study (3.43 ± 0.34). The differences in results may be associated with variations in the processing technology used to obtain the fermented product, where in the present study the producers of this drink usually add water after fermentation to increase the amount of ucanhi produced.

 

The low acidity of the fermented products tested in the present study is a desirable characteristic in terms of food safety and sensory for the adult consumer, however, for children consumption, it can be a denial factor.

 

Quantification and identification of LAB

 

Figure 1 illustrates the quantification of LAB by each fermented food. All traditionally fermented foods under study had low LAB counts, ranging from 2.54 to 4.23 Log CFU/g or ml. Specifically, the counts were 2.54 ± 0.57; 3.82 ± 0.73; 4.07 ± 0.41; 4.23 ± 1.09 Log CFU/ml or g, for ucanhi, rhali, maheu and massi, respectively.

 

The results regarding the LAB counts obtained in the present study for rhali are below those reported by Huch et al. (2008) who obtained LAB counts variable from 2 × 102 to 6 × 103 CFU/g during the evaluation of the use of species of Lactobacillus for initiation of cassava fermentation in Gari production. In turn, Penidoa et al. (2018) reported LAB counts above those observed in the present study, ranging from 5.8 to 7.9 Log CFU/g, in samples of fermented cassava flour using natural microflora for 56 days. The difference in results between the two studies may be associated with the technology used to process this product in Mozambique, where the fermentation process usually takes 3 to 6 days, followed by the roasting step of the fermented pulp.

 

 

Similarly to the rhali, the results of the LAB counts obtained in the massi are below the results reported by Beukes et al. (2001) where they observed that LAB counts ranged from 5.7 to 9.1 Log CFU/ml in amasi produced in South Africa.

 

Nyambane et al. (2014) obtained LAB mean counts higher than those reported in the present study, varied from 7.86 Log CFU/ml to 8.32 Log CFU/ml in massi samples processed in gourds and plastic containers, respectively. This fact can be explained by the existing variations in the processing technologies used in the two studies in reference for this traditionally fermented food, as is the case of time (which varies from 1-3 days) for the present study and 4 days for the study of Nyambane et al. (2014), the environment fermentation temperature of the present study and the environment  temperature (18 to 32ºC) in the study by Nyambane et al. (2014).

 

The LAB counts obtained for the maheu samples are below the results reported by Simatende (2016), which found that LAB count varies from 8 - 10 Log CFU/ml, during the characterization and diversity of the microflora present in the emahewu produced in Swaziland, results that approximate twice the LAB counts obtained in the present study. In the same order of ideas, Mashau et al. (2020), when assessing the evaluation of the shelf-life extension and sensory properties of mahewu - a non-alcoholic fermented beverage, by adding Aloe vera powder obtained LAB counts ranging from 3.0086 to 7.7559 Log CFU/g.

 

This fact may be explained because of the addition of warm water after fermentation of the maheu and the reduction of the fermentation period (from 3days to 1 day) during the process of preparation of the samples used in this study. This statement differs from the ones reported by Simatende (2016), whose technology of production does not include the addition of water and long fermentation (2-3 days in summer and up to 5 days in winter). The low pH of mahewu contributed for the increase of lactic acid microfola during fermentation allowing the growth of LAB, which resulted in competing microorganisms being inhibited.

 

The results obtained in ucanhi are similar to the results reported by Nyanga et al. (2007), who verified that the LAB count in the marula pulp (Sclerocarya birrea) ranges from  2.91  to 2.99 Log UFC/g. Phiri (2018) found that the LAB count ranges from 2.27 × 103 to 1.57 × 105 CFU/ml. The similar results can be justified by the similarity in the processing technology described in the literature by the authors (Nyanga et al., 2007; Phiri, 2018) and those mentioned in the present study. Although, LAB have a beneficial effect and some strains have a probiotic effect, where a high concentration of these in food must be guaranteed.

 

 

From the isolated pure colonies, the following LAB species were identified: Lactobacillus plantarum, L. fermentum, L. collinoides, Leuconostoc mesenteroides subsp mesenteroides/dextranicum 2 and Pedicoccus pentosaceus. The summary of the morphological characteristics of each identified LAB is shown in Table 2. However, it was still observed the growth of molds of the genus Fusarium in rhali and Mucor in massi.

 

LAB species with probiotic potential described in the literature on similar products in different African countries (Beukes et al., 2001; Jans et al., 2017; Kayitesi et al., 2017) are L. plantarum, L. fermentum, Leuconostoc mesenteroides ssp. mesenteroids and Lactococcus. Lactobacillus plantarum as a probiotic has been described by some authors (Nyanga et al., 2007; Nyambane et al., 2014). This LAB is homo-fermentative and ferments lactose to produce lactic acid as its main metabolic product.

 

Simatende (2016), describes L. plantarum as a typical biota of non-alcoholic beverages spontaneously fermented with corn and soy, playing a key role in defining the attributes of these products. Leuconostoc mesenteroides ssp. mesenteroids during spontaneous corn fermentation can inhibit the growth of Aspergillus flavus (Rahmawati et al., 2013).

 

Yeast quantification and identification

 

All  traditionally   fermented  foods  under  study  had  low yeast counts, around 2.24 to 3.63 Log CFU/g or ml, ranging from 2.24 ± 0.43; 2.92 ± 0.37; 3.22 ± 0.87 and 3.63 ± 0.55 Log CFU/g or ml for ucanhi, massi, maheu and rhali, respectively. It was also observed the growth of molds of the genus Fusarium in a sample of rhali. Figure 2 illustrates the morphology and quantification of yeasts by fermented food.

 

The obtained results referring to yeast counts for dough samples are below the results reported by Nyambane et al. (2014), obtained yeast counts that ranged from 6.65 to 7.62 Log CFU/ml and from 5.50 to 6.65 Log CFU/ml in dough samples processed in gourds and plastic containers, respectively. The high acidity, allied to the high environment, temperature verified in the sampling area, can constitute inhibitory factors to the multiplication of yeasts in these products.

 

For the case of maheu, the results obtained from the yeast counts in the present study were similar to those reported by Rahmawati et al. (2013), who obtained variable values of 3-5.5 Log CFU/g in maheu. The results obtained in ucanhi are similar to the results reported by Nyanga et al. (2007) and Phiri (2018) which verified that the yeast count varies from 2 to 6 Log CFU/ml.

 

Regarding rhali, the results obtained are below those reported by Penidoa et al. (2018) which reported yeast counts from 1.7 to 7.8 Log CFU/g in fermented cassava flour. This factor may be associated with the fact that the samples used in this study undergo the process of removing excess moisture, a long fermentation time of 3 to 6 days and heat treatment (roasting over an open fire).

 

The identification of yeasts at the species level confirmed that the most prevalent are: Candida albicans, Cryptococcus humicola, Rhodotorula mucilaninosa 2, Saccaromyces cerevisiae, Candida tropicalis, Stephanoascus ciferri, Trichosporan mucoides, Candida dubliniensis, and Candida famata. The summary of the morphological characteristics of each identified yeast is illustrated in Table 3.

 

 

 

 

Yeast species with probiotic potential described in the literature for traditionally fermented products evaluated in different African countries similar those obtained in the present study include: Candida albicans, Candida famata, S. cerevisiae, Rhodotorula mucilaninosa (Nyanga et al., 2007; Rahmawati et al., 2013; Nyambane et al., 2014). According to Nyambane et al. (2014), S. cerevisiae has been associated with the production of alcohols and other aromatic compounds, stimulation of LAB, improvement in nutritional value and inhibition of undesirable microorganisms.

 

Rahmawati and collaborators (2013), by isolating and identifying microorganisms during spontaneous corn fermentation (maheu), described Candida famata as having high glucoamylase activity, producing biomass and exhibiting lipolytic and proteolytic activity. Nyanga et al. (2007) reported that Rhodotorula mucilaninosa occurs as a natural flora in marula fruits, when the fruit matures, fermentation occurs naturally as a result of its presence, fermenting sugars into alcohol.

 

Contrary to expectations, the opportunistic pathogen C. albicans was isolated in some samples of massi and rhali. Nyambane and collaborators (2014) stated that the presence of C. albicans is of concern, as it can cause superficial, localized and/or systemic infections in humans. The presence of this species in the samples evaluated in this study requires additional and in-depth investigations and may be is an indicator of deficient hygienic processing and/or commercialization practices.

 

Microbiological safety of traditionally fermented foods

 

The growth of green colonies with metallic shine on EMB Agar was verified in 4 samples of rhali, 17 samples in massi and 7 samples in maheu. These samples were also positive to the Indole test, confirming the presence of E. coli in the products under analysis.

 

As described by Mwale (2014) and Kayitesi et al. (2017), the presence of pathogenic microorganisms occurs due to the use of primitive methods of production of fermented foods, as well as non-compliance with good hygiene and processing practices. Nyambane et al. (2014) related the high prevalence of Enterobacteriaceae to the presence of acid-resistant E. coli strains and coliforms.

 

Molds and yeasts were observed in some samples of traditionally fermented foods, 5 in rhali, 18 in maheu and ucanhi, and 15 in the massi. Traditionally fermented foods presented mean count of mold colonies around 2.18 to 4.29 Log CFU/ml or g, ranging from 2.18 ± 0.15; 3.50 ± 0.42; 3.69 ± 0.89; 4.29 ± 0.45 Log CFU/ml or g for ucanhi, maheu, rhali and massi respectively. The growth of molds was verified in samples of massi cultivated in MRS having 3.09 ± 0.60 Log CFU/ml and ucanhi samles grown on YPD agar having 1.40 ± 0.15 Log CFU/ml. Figure 3 illustrates the quantification of molds by fermented food.

 

In samples that showed growth of molds and yeasts, some  yeasts  were  identified.  Mold   colonies   such   as Fusarium spp on rhali have also been identified; Mucor in massi; Paecilomyces fumosoroseus in the maheu; Geotrichium candidum and Sporotrichum in b. canhú. The summary of the morphological characteristics of each identified yeast is illustrated in Table 4. 

 

 

The presence of these microorganisms may be due to non-compliance with good hygiene practices in processing and especially in marketing. It is well known that fungi generally withstand extreme conditions better than bacteria and are found in foods with a low pH (with acidity up to 3.5). Some authors (Simatende, 2016; Phiri, 2018) have identified Geotrichum capitatum as part of the normal microflora of the marula.

 

As rhali is a product with low water activity and often exposed to environmental contaminants during fermentation, a certain growth of molds of the Fusarium species was expected, which is generally associated with inadequate storage of the final product. This fact is alarming in terms of public health, since rhali is a product for direct human consumption.


 CONCLUSION

According to the results obtained, it can be concluded that traditionally fermented foods under this study, are quite acidic and have relatively low LAB and yeast counts. In the four products studied, new LAB species and yeasts with probiotic potential were identified. In maheu, Lactobacillus collinoides, Stephanoascus ciferri, Trichosporan mucoides, Cryptococcus humicola and Candida dubliniensis; in ucanhi, Pedicoccus pentosaceus, Cryptococcus humicola and Candida famata; in rhali, Trichosporon mucoides, Cryptococcus humicola, Stephanoascus ciferri, Saccharomyces cerevisiae and Candida albicans; yeasts in massi, Cryptococcus humicola, Rhodotorula mucilaninosa 2 and Candida tropicalis. For all traditionally fermented foods, microorganisms of food safety concern were isolated, namely E. coli, Fusarium sp, Mucor,  Paecilomyces fumosoroseus, Geotrichium candidum and Sporotrichum, showing a risk to the health of the consumer.


 ACKNOWLEDGMENTS

The authors would like to thank the National Research Fund (FNI) for funding the PROBIO project that contributed to this work and to Chalmers University of Technology for their cooperation in identifying LAB and yeast isolates.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



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