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Starters are of great economic importance to our society because of their use in various sectors. It is then important to maintain their viability during the manufacturing process and storage. Freeze drying is one method that is commonly used to preserve bacteria. In this study, various flours were compared with two known cryoprotectants, mannitol, and saccharose, for their cryoprotective potential for acetic acid bacteria. A strain of Acetobacter pasteurianus was grown in YEPG broth and centrifuged. The pellet was then collected and mixed with 20% saccharose, 20% mannitol, a combination of 10% mannitol with corn flour, sorghum flour, millet flour, rice flour, soy flour, a combination of 20% saccharose with maize flour, sorghum flour, millet flour, rice four, soy flour. The freeze-dried starters were stored at room temperature (25 °C), and viability was checked weekly after freeze drying for a storage period of 5 weeks. The results show that sorghum flour, soy flour, and maize flour had survival rates in the order of 73%–78% when used alone. Soy flour alone helped maintain the viability of the strain above 50% during storage for 4 weeks. Soy flour could be used as new potential support for the freeze-drying of Acetobacter pasteurianus. This could help solve one of the many problems in the cocoa culture.

Introduction

Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic bacteria and are usually found in nature on fruits, cereals, herbs, etc. They have an ability to oxidize different kinds of alcohols and sugars into vinegar, cellulose, sorbose, gluconic acid, etc., [1]. Moreover, they are very important for the biotechnology, pharmaceutical, and food industries and are extensively used as probiotic products and starter cultures to improve traditional fermentations, especially cocoa fermentation [2]. Indeed, cocoa fermentation is a spontaneous and random process, leading to variable cocoa beans’ quality. Hence, it is important to standardize the process using microbial starters containing some of the key players, particularly acetic acid bacteria. Acetic acid bacteria play an important role in the fermentation process. They are responsible for the conversion of ethanol and, in some instances, lactic acid and mannitol into acetic acid. The acid then deeply penetrates the cotyledon, increasing the acidity inside the beans. Next, follows a cascade of reactions that lead to the formation of aroma precursors of cocoa [3]. Previous studies carried out in our laboratory and in other countries have isolated and selected acetic acid bacteria starters capable of improving and standardizing the cocoa fermentation process [4]–[7]. These starters need to be put into a much more suitable form to facilitate the process by cocoa farmers. There are other problems, especially a lack of electricity, which will prevent them from having a cold room to maintain starter viability. The most suitable form of conserving these starters is in the powdered form which can be achieved through freeze-drying. Acetic acid bacteria are generally freeze-dried with mannitol. Mannitol is a polyalcohol widely used in lyophilization because of its properties as a bulking agent. Other cryoprotectants like saccharose can also be used as well. They help protect the lipid bilayer from collapsing under stresses during freezing by forming a glassy state inside and outside the lipid bilayer membrane [8].

These cryoprotectants (mannitol, saccharose) are not easily accessible, and they are expensive in Côte d’Ivoire, from where the need to find inexpensive and easily accessible elements in order to bring the technology of using starters culture viable in cocoa farming.

The main aim of this study is to find among local flours those capable of being used as cryoprotectants or carrier material to freeze-dry Acetobacter pasteurianus starters culture to facilitate their use by cocoa farmers in Côte d’Ivoire.

Materials and Methods

Materials

The bacteria strain used in this study was isolated from fermenting cocoa from Côte d’Ivoire [3], [9]. The bacterial strain of Acetobacter pasteurianus was preserved in YEPG broth (1% yeast, 4% ethanol, 1% peptone, 1% glucose) broth with 20% glycerol at −80 °C. Five (5) flours (corn flour, sorghum flour, rice flour, millet flour, soy flour) purchased from a supermarket in Cocody in the District of Abidjan were also used.

Methods

Culture of Acetobacter pasteurianus Starters for Freeze-Drying

The selected starter Acetobacter pasteurianus was revived for 24 h in YEPG broth and then replicated on YEPG agar plates. The starter strain was then incubated at 37 °C for 48 h. A pure colony was used to prepare a dense suspension with 120 ml of YEPG broth. The cultures were left for 5 days at 37 °C in an oven.

Lyophilisation of Acetobacter pasteurianus Starters

The acetic acid bacteria culture left in YEPG broth was centrifuged at 12000 × g for 5 m at −4 °C in a refrigerated centrifuge (Laboao, China). The pellets were washed twice using saline solution (0.9% NaCl) and harvested. The flours used in this study were prepared to obtain a final concentration of 4%. About 2 g of flour was dissolved in 50 ml of distilled water. The mixture was heated to a final temperature of 70 °C–80 °C for 20–30 m with constant stirring. The mixture was then cooled to around 30 °C–40 °C [10]. Two cryoprotectants (mannitol and saccharose) were used. Some of the compositions contained 20% saccharose and/or 20% mannitol. The different compositions of the essay used were summarized in Table I. Next, the mixtures (essay) were frozen at −60 °C for 6–24 h and freeze-dried for 36 h at 1 Pa and −45 °C in a freeze-dryer (Laboao, China).

Essay Flour (ml) Saccharose (ml) Mannitol (ml) Physiological saline (ml)
1 0 0 0 25
2 0 0 5 20
3 0 5 0 20
4 2.5 millet flour 0 0 22.5
5 2.5 maize flour 0 0 22.5
6 2.5 soy flour 0 0 22.5
7 2.5 rice flour 0 0 22.5
8 2.5 sorghum flour 0 0 22.5
9 2.5 soy flour 0 5 17.5
10 2.5 sorghum flour 0 5 17.5
11 2.5 rice flour 0 5 17.5
12 2.5 millet flour 0 5 17.5
13 2.5 maize flour 0 5 17.5
Table I. Different Compositions of Cryoprotectant Used for Freeze-Drying

Determination of Survival Rate of Acetobacter pasteurianus Starters after Freeze-Drying

Serial dilutions’ method described by [11] was used to determine the survival rate of Acetobacter pasteurianus onto YEPG agar plates in microbial culture made in Section 2.2.1. After freeze drying, an amount of 0.1 g of freeze-dried bacterial powder was diluted in 4 ml of physiological saline for each composition of the starter (essay). The samples were incubated at 37 °C for 2 h. Next, serial dilutions of each sample incubated were prepared, and a volume of the cell suspension was inoculated uniformly onto the YEPG agar plate. The plates were kept at 37 °C for 72 h. Cell viability was determined using a standard count method on YEPG agar medium. The dilution times and the corresponding number of bacteria were recorded. The average of the three plates was used for the number of bacteria per dilution, in which the plate with a bacteria count of 30–300 CFU and no spreading colony growth was selected. The survival rate of Acetobacter pasteurianus after freeze-drying process was expressed according to the method of [12]. Cell viability obtained for each (essay) was expressed as Survival Factor (SF) in percentage, calculated using the following equation:

(1)SF=1−(logCFUbefore−logCFUafter)logCFUbefore×100where

CFUbefore–CFUml−1 × total volume culture (ml) before the freeze − drying process

CFUafter–CFUg−1 × total weight of the dry bacterial sample (g)

Determination of Acid Production Capacity of Acetobacter pasteurianus Starters after Freeze-Drying

After freeze drying, the samples were revived in physiological saline. The cell count was determined using Thomas’ cell. The charge of the samples was fixed at 105 cell/ml to inoculate a flask containing 15 ml solution of YEPG broth. Each sample was assessed before freeze-drying for titratable acidity. Titratable acidity was determined by titrating 5 ml of each sample with 0.1 N sodium hydroxide (NaOH) solution. Acid production (Pa) was determined by the following formula:

(2)Pa=Nb×Vb×1000×MVawhere

Va–Volume of the sample (ml)

NbNormality of NaOH (ml)

VbVolume of NaOH (ml)

M–Molar mass of acetic acid

Determination of Viability of Freeze-Dried Acetobacter pasteurianus Starters During Storage at Room Temperature

The bacterial count method, as used in Section 2.2.3, was used for the evaluation of cell viability over time. The bacterial count and survival rate were calculated using the method of Chen et al. [13]:

Bacterial count (CFU/ml)—average number of colonies in 3 replicates of the same dilution × dilution times.

Survival rate (%)—viable cell number after freeze-drying/viable cell number before freeze-drying × 100%

Statistical Analysis

All experiments were repeated at least three times, and the raw data generated were expressed as the mean±standard deviation. The calculations and figures were performed using Excel 2016. Descriptive statistics were used to analyze survival rates and performance of lyophilized cultures. One-factor analysis of variance (ANOVA) was used to compare means. Means were separated by Tukey’s error rate multiple comparison test using XLSTAT software, and differences in means were considered statistically significant at p < 0.05.

Results

Survival Rate of Acetobacter pasteurianus in Different Cryoprotectants after Freeze-Drying

The Table II below shows the results of the survival rate of Acetobacter pasteurianus in thirteen different cryoprotectant compositions, namely maize flour, sorghum flour, millet flour, rice flour, soy flour, mannitol, and saccharose. In general, two main groups of survival rates were observed. The first is observed when the following cryoprotectants are used: soy flour/mannitol, sorghum flour/mannitol, rice flour/mannitol, saccharose alone, millet flour, millet flour/mannitol, maize flour/mannitol, sorghum flour, mannitol, soy flour with values ranging from 73.27% ± 2.92% to 78.68% ± 4.66%. They all had statistically identical values. Lower values were obtained in the second group when the following cryoprotectants were used: Acetobacter pasteurianus alone without any additives, maize flour, and rice flour with values ranging from 63.63% ± 3.21% to 66.20% ± 1.46%.

Cryoprotectant Survival rate (%)
Soy flour/mannitol 78.68 ± 4.66a
Sorghum flour/mannitol 76.55 ± 3.07a
Rice flour/mannitol 75.98 ± 1.46a
Saccharose 75.97 ± 3.59a
Millet flour 75.89 ± 5.18a
Millet flour/mannitol 75.80 ± 1.59a
Maize flour/mannitol 75.28 ± 1.96a
Sorghum flour 75.25 ± 0.98a
Mannitol 73.38 ± 4.97a
Soy flour 73.27 ± 2.92a
Acetobacter pasteurianus 66.20 ± 1.46b
Maize flour 64.28 ± 2.57b
Rice flour 63.63 ± 3.21b
Table II. Survival Rate of Acetobacter pasteurianus in Different Cryoprotectants After Freeze-Drying

Determination of Relative Titratable Acidity of Acetobacter pasteurianus in Different Cryoprotectants after Freeze-Drying

The Table III below shows the relative titratable acidity after freeze-drying of different cryoprotectant formulations. With the different cryoprotectants used, we have values between 48.95% ± 1.80% and 84.09% ± 2.71%. It was observed that maize flour/mannitol had the highest relative titratable acidity of 84.09% ± 2.71% after freeze-drying compared to all the other formulations. The acidity of the mixture without cryoprotectant was similar to that of soy flour combined with mannitol. Cryoprotectants with maize flour, sorghum flour, soy flour had statistically significant percentages of acidity as the one without cryoprotectant. These acidity values ranged from 72.43% ± 3.98% to 74.51% ± 2.43% and were higher than those of saccharose (60.69% ± 3.87%) and mannitol (48.95% ± 1.80%).

Cryoprotectant Relative titratable acidity (%)
Mannitol 48.95 ± 1.80a
Saccharose 60.69 ± 3.87b
Rice flour/mannitol 61.80 ± 4.20bc
Rice flour 63.88 ± 2.40bcd
Millet flour 65 ± 4.33bcd
Sorghum flour/mannitol 68.19 ± 4.45cde
Millet flour/mannitol 70.34 ± 5.69de
Soy flour 70.34 ± 5.69de
Maize flour 72.43 ± 3.98e
Sorghum flour 72.43 ± 3.98e
Acetobacter pasteurianus 73.47 ± 2.76e
Soy flour/mannitol 74.51 ± 2.43e
Maize flour/mannitol 84.09 ± 2.71f
Table III. Titratable Acidity of Various Cryoprotectants of Acetobacter pasteurianus after Freeze-Drying

Determination of Relative Survival Rate over Time in Different Cryoprotectants after Freeze-Drying

Fig. 1 shows lyophilization of Acetobacter pasteurianus in the presence of different cryoprotectants to study their effect on viability over time. Viability was expressed as a percentage of relative growth. Initially, the viability rate of all cryoprotectants was expressed at 100%. Overall, a decrease in viability growth was observed for all cryoprotectants over the five (5) weeks of storage, particularly during weeks 3 and 4 of storage. All twelve (12) preparations containing cryoprotectants had a much higher survival rate than the sample without any cryoprotectant. The sample without cryoprotectant showed 100% loss of viability after 3 weeks of storage. The highest viability growth was measured for soy flour (80.31% ± 0.20%) after 1 week of storage, which represents a loss of only 19% throughout the storage period. Saccharose provided the greatest protection during the 5 weeks of storage, with a loss of about 45% after 4 weeks of storage. After five (5) weeks of storage, all samples experienced 100% viability loss.

Fig. 1. Relative survival rate of Acetobacter pasteurianus in the presence of different cryoprotective agents for five (5) weeks storage. Experiments were performed at least in three independent replicates. Error bar shows the standard deviation.

Discussion

The main aim of the study was to find alternative cryoprotectants to be used in the freeze drying of Acetobacter pasteurianus. Freeze-drying is a widely used method for the long-term preservation of microorganisms in dehydrated form [14]. However, not all microorganisms are able to withstand this drying process. To reduce cell death, cryoprotectants can be used during this process [15]. This study showed that the survival rate of Acetobacter pasteurianus after freeze-drying was more than 50% for the 12 formulations used.

When millet flour, sorghum flour, and soy flour were used alone, they gave similar survival rates after freeze-drying as mannitol and saccharose, which are generally used for freeze-drying bacteria [16]–[18]. This suggests that these three flours are composed of elements with cryoprotective functions. Indeed, sugars are among the elements used as cryoprotectants [19], and most of these flours are rich in carbohydrates [20]. However, there are few studies reporting on the use of cryoprotectants in the freeze drying of Acetobacter pasteurianus. As expected, a loss of viability (66%) was observed for lyophilised Acetobacter pasteurianus cells without cryoprotectant. The values obtained are in agreement with Jagannath et al. [21] those who reported viability of lactic acid bacteria using different cryoprotectants of 67%–70% after freeze-drying.

Regarding storage, our results show that of the three flours (sorghum flour, soy flour, maize flour), only soy flour maintains the strains’ viability at over 50% after 4 weeks of storage. These results were similar to those obtained with saccharose and were 20% lower when mannitol was used as the carbon source. This could be due to the fact that soy flour is rich in protein (about 35%–50%) and low in reducing and total sugars compared to the other flours [22]–[24]. Meanwhile, millet flour/mannitol, maize flour, rice flour/mannitol, sorghum flour/mannitol, and millet flour formulations maintained their viability after 3 weeks of storage. Sorghum flour, soy flour/mannitol, rice flour, and mannitol remained viable after 2 weeks. As for the formulation with the strain alone, it retained its viability after only one week of storage. This value is in accordance with Maneesri et al. [25] those who found a loss of viability in the starter powder after one week of storage in Acetobacter aceti. Similar results have been reported in the literature suggesting that the stability of starter powder stored at room temperature decreases quickly in 1 month of storage. Viability during storage can be caused by many factors, such as the strain of the microorganism. It can also be caused by cell damage at different sites of the cell membrane during osmotic stress. The presence of cryoprotectants helps to maintain good viability by changing the viscosity within the bacterial cell, by participating in direct intermolecular interactions, and by modification of the water activity within the bacteria [16]. In addition to the use of cryoprotectants, microorganisms tend to survive poorly at relatively higher temperatures due to the increased rate of metabolic activities and other chemical or enzymatic reactions, such as lipid oxidation, which may also affect cell survival [26]. Mannitol is one of the most studied cryoprotectants. It is a polyalcohol with four hydroxyl groups arranged along one side. This structure favours interactions with the membrane bilayer. Therefore, mannitol helps to stabilize the phospholipid bilayer and membrane proteins during cellular dehydration and freezing, thus improving cell survival during conservation [8]. This may explain why better results were obtained with mannitol alone and in combination with other carriers. The protective effect of saccharose was investigated by Jawan et al. [27] when they studied the effect of different types (monosaccharides, disaccharides, sugar alcohol, complex media) and concentrations (5%, 10%, and 20%) of lyoprotectants on cell viability and antimicrobial activity of freeze-dried Lactococcus lactis Gh1. They found that 15% or 20% sucrose combined with a high bacterial concentration gave the best stability. This result is consistent with the one obtained, which could explain why saccharose was the best cryoprotectant preserving cell stability as well as high acidification capacities.

Assessing titratable acidity is necessary to evaluate the ability of the freeze-dried strains to recover their acidification activity after dehydration. Finally, we observe that the acidification capacity of Acetobacter strains after freeze-drying depends on the different protective elements used, while the freeze-dried Acetobacter pasteurianus strain shows a reduction of more than 30%. This suggests that freeze-drying may have an impact on the genes involved in the production of organic acids by Acetobacter. In addition, the presence of cryoprotective elements reduces the positive or negative effect of freeze-drying [28], [29]. This result contradicts some authors who have shown that freeze-drying has no effect on the genes responsible for the synthesis of primary or secondary metabolites [30].

Conclusion

Overall, the effect of various flours alone and in combination with known cryoprotectants (mannitol and saccharose) were evaluated on the survival rate and performance parameters of freeze-dried Acetobacter pasteurianus in this study. The results concluded that the flours behaved differently after freeze-drying and during storage at room temperature and in terms of acid production. Among the flours, soy flour had the highest survival rate after freeze drying and good storage stability for four weeks at ambient temperature. It is a good alternative to known cryoprotectants such as mannitol and saccharose.

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