Involvement of Bacillus Species in the Understanding of the Softening Process of Safou Pulp (Dacryodes Edulis H.J.Lam)

of in the Understanding of the Process of Abstract For the last twenty years, Biotechnology, including techniques for preserving of traditional fermented foods, has grown considerably. Safou, some fruit from Dacryodes edulis, is widely consumed in Africa but poses conservation problems because of its very rapid softening. In the Republic of Congo, a fermentation technique has been developed in order to preserve fruits. This work aims to study the bacteria of the genus Bacillus involved in the softening of Safou. Using classical microbiological techniques, we estimated the number of bacteria to be 2.5±0.7. 102 FCFU/g from the softened safous and at 4±0.8. 106 CFU/g from the non-softened safous. By using PCR multiplex of fibE gene, Bacillus subtilis and Ballus safensis have been mostly ientified and strains have shown degradation percentages of pectin, amylose, proteolytic up to 55%, 55% and 63 %, respectively. 45% of isolates including Bacillus subtilis and Bacillus safensis can secrete pectinases, amylase and proteases. The assessment of Penetrometry index showed that B. subtilis and B. safensis are able to contribute to the softening of african pears.


Introduction
Dacryodes edulis occurs naturally in the countries bordering the Gulf of Guinea. It is croping from Sierra Leone east to Angola along the Atlantic and further inland to Uganda. It has also been reported from northern Zimbabwe. Safou or African pears are typical fruits can be produced by Dacryodes edulis crop. The culture of Dacryodes edulis has spread an attracting growing interest and is currently in strong progression in the continent of Africa. In Africa population this fruit is well appreciate [1,2]. Safous have been found not only in Africa countries, but also it is exported in Europe including France, Nederland, Germany and Belgium. More and more the demand of safou on the Congolese market has become strong. But the local production is weak and african pear is still a seasonal fruit in the Republic of Congo. One of the major and worrying problems is the preservation of the safou. The post-harvest rot diseases of fruits of the African pear are one the economic concern in Africa [3,4]. In order to preserve fruits, the congolese people have developed a tradi tional strategy consisting in fermenting the safous and drying them.
Most of the scientific research carried out on D. edulis has focused on the nutritional value of african pear pulp and oil extraction processes [5][6][7]. By drafting this paper no studies have been forcused to understand the fermentation process of safous. In addition, endogenous knowledge states that the safou once softened can be eaten or dried for future preservation. The microorganisms involved in the softening of african pear have not yet been studied. This study aims to speciffically the understanding in the role of Bacillus species in the softening of the traditional process of Safou preservation.

Isolation of Microorganisms
The mixture was heated in a microwave for 3min until agarose was completely dissolved and then cooled in a water bath at 40°C. Then 10ml of skim milk was added to the mixture. After homogenization, the mixture was poured into the Petri dishes. Once solidified, wells were carefully and aseptically generated into the gels. A volume of

Amylolytic and Pectinolytic Activities
To assess the amlylolytic, pectinolytic activity of isolates, an overnight culture in LB medium was carried out on a Petri dishes. safensis, and B. atrophaeus [8]. Briefly, extraction and purification of isolate genomic DNA were performed according to the Nucleo Spin Microbial DNA (Macherey-NAGEL) kit. Briefly, isolates were grown in 5ml LB broth for 24h at 37°C with stirring. The DNA purity was assessed by electrophoresis on agarose gel and by the ratio of optical densities 260/280nm. 5μl of each amplification product was mixed with 2μl of loading buffer (BIOKE). Mixtures were subjected to electrophoresis on 1% agarose gel (w/v). The 10kb 2-Log (BIO-KE) was used as a molecular weight marker.

The Influence of Identified Bacteria in the Softening
The penetrometer resistance is an effective and reliable method for evaluating safou puple softening. A mecanic penetrometer has been used to introduce in softened African pears. Five overnight culture corresponding to NIc3, Nic32, Nic37, Nic39 and Nic45 were done. The pure culture has been assessed to reach 0.8 to 1 of D.O. Safou have been first washed with aseptic water and sterilized with 70% and 90% of ethanol. The aseptic safous were immersed for 5 to 10min in each of the cultures and in the culture put together. The samples were stored at room temperature to follow the softening process. The penetrometry indices were calculated during the fermentation process. The values were established according to the texture of the softening safou. A score of ten (10) was associated with the safou who's the penetrometer completely has been penetrated. A score of five (5) was associated with the safou who's the penetrometer has not been completely penetrated. Zero (0) was associated with the safou puple whose peak strength and maximum stress were not allowable.

Molecular Identification of Isolates
A strategy has been implemented in our laboratory as part of a previous study. We therefore sought to identify by using this avec been associate to Bacillus safensis (Figure 2). Nothing has been amplified Nic 3, 32, 40, 42, 43 and 45.

Discussion
In this work we have worked on ripe fruits of safou collected in local markets in Brazzaville. One of major trouble shooting of safou comes to the short duration of natural softening. The post-harvest losses by natural softening of safou pulp have been studied [6]. This work aimed to isolate and to molecularly identify the microorganisms like Bacillus and to understand the enzymatic activities in the softening of safou. The isolation from ten (10) softened safou fruits showed a low enumeration rate which was around 2,5±0.7. 10 2 Colony Forming Unit per gram of material. This low rate would be due to the low specific activity of the water. Isolation of non-softened african pear has been compare with the softened safou. As result the numeration was about 4±0.8 . safensis. Our inexpensive and rapid method has the advantage of avoiding gene sequencing steps [8].
Bacteria such as Bacillus thuringiensis have also been identified by a direct approach using primers which hybridize specifically on the cry gene of course without going through sequencing [9]. Another study on yeasts had shown that, thanks to the direct PCR approach, it was also possible to directly identify yeasts such as Saccharomyces cerevisiae, S. arboricola, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. pastorianus. All this by targeting specific genes belonging to these species [10].
Fruit softening is a major issue of multiple cellular processes, including extensive remodeling of cell wall structure [11]. The ripening of the fruits is a complex event and some molecular events occurring during softening of strawberry fruit have been documented [12]. It is obvious to admit that ripening of fruits like safou is also identified by the secretion of a range of pectin degrading enzymes in the cell wall, and changes in the structure of pectin could result from their synergistic combination with the physiology of the cell [11] and enzymes secreted by Bacillus. The flesh of the fruit is softened by enzymes that break down cell walls. The natural action of these enzymes is dependent on the temperature [13] and would be accelerated by the enzymes produced by bacteria of the genus

Bacillus.
The genus Bacillus is known for its ability to produce extracellular enzymes such as amylases pectinases, proteases [14][15][16][17]. In this study we demonstrated the correlation between the secretion of enzymes and safou softening. In the context of this work some safou. The enzymatic activities carried out have shown that bacteria of the genus Bacillus are able to secrete lytic enzymes produced during the softening that could contribute to the degradation of cell wall structures. It has been documented that softening is a hallmark of fermentation in most modern or traditionnal fermented food [18][19][20][21].

Conclusion
There is a bright future for african pear generally appreciated for its health benefits. In view of the afore informations, there is an involvement of microorganisms in the softening of the pulp of safou. The softening process is orchestrated by the coordinated secretion of enzymes such as proteases, pectinases and proteases by the genus Bacillus belonging to the phylogenic group I. This work seems to be very important for the valorization of the african pear.
Several enzymatic activities can be studied, such as lipolytic and cellulosic activity, which may also help explain the softening of safous. We plan to continue this work by studying the protein profile and molecular identification of all cultivable flora.