Use of Sweet Potato as a Component in the Development of Laboratory Medium for the Cultivation of Lactobacillus.sp

doi:10.21203/rs.3.rs-3989756/v1

Download PDF

Research Article

Use of Sweet Potato as a Component in the Development of Laboratory Medium for the Cultivation of Lactobacillus.sp

https://doi.org/10.21203/rs.3.rs-3989756/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Due to its abundance, low cost and sustainability, sweet potato, a widely cultivated tuberous root rich in carbohydrates and essential nutrients, presents an intriguing prospect as a potential growth medium for Lactobacillus.sp. In the present study, we investigated the feasibility and efficacy of sweet potato-based medium for supporting the growth of Lactobacillus.sp bacteria. The sweet potato medium was supplemented with 2, 8, or 14 g/L of plant- based nitrogen sources (X-Seed Nucleo Max, X-Seed KAT, and NuCel 780 MG) to form SPM1, SPM2, and SPM3 respectively. Seven Lactobacillus.sp strains were used in this study, and a Lactobacilli MRS growth medium was used as a control. The growth pattern of tested Lactobacillus.sp strains grown in SPM2 and SPM3 was similar or higher than that in MRS. The population of Lactobacillus.sp strains reached an average of 7.94 ± 0.11, 8.38 ± 0.11, and 8.57 ± 0.12 at 12 h of incubation for MRS, SPM2, and SPM3, respectively. The buffering capacity of SPM2 and SPM3 was significantly (p < 0.05) higher than that in MRS, and the growth in SPM1 was lower than that of MRS. These findings demonstrate that SPM2 is suitable for the growth of Lactobacillus.sp strains and could be used as an alternative medium.

Lactobacillus.sp

sweet potato

plant-based nitrogen

According to [1], the global market for lactic acid bacteria (LAB) was estimated at $ 3.1 billion in 2022 and is projected to grow at a compound annual growth rate of 8.0% from 2023 to 2030. LAB is used extensively in many food applications such as meats, vegetables, dairy products, beverages, and other fermented products [2]. High cell mass production of LAB has become increasingly important as the food industry has developed an interest in LAB not only as a starter culture but also as a valuable probiotic supplement for human health benefits [3, 4]. The supplementation of culture growth media with various nutrients is crucial for the optimal growth of LAB strains. LAB requires a diverse range of nutrients, including amino acids, peptides, nucleic acid derivatives, fatty acid esters, minerals, vitamins, and buffering agents, in addition to carbohydrates [5]. Nitrogen is also an essential component of culture media, and traditional nitrogen sources used in LAB medium include peptone, yeast extract, and beef extract [6]. These nitrogen sources are rich in free amino acids, peptides, and proteins, which serve as nitrogenous compounds for bacterial growth and protein synthesis [7, 8]. However, these ingredients can be expensive and contribute significantly to the overall cost of the media. To address this, researchers have explored alternative options by investigating the potential of low-cost food items and agricultural wastes as substitutes for expensive nitrogen sources [9, 8]. By utilizing readily available and inexpensive materials, it is possible to reduce the production costs of LAB culture media without compromising the growth and viability of the bacteria. There are several alternative media formulations that have been developed for the cultivation of LAB. These alternative media aim to reduce costs by replacing or reducing the reliance on expensive components while still providing the necessary nutrients for LAB growth [10]. Here are a few examples of alternative media used for LAB cultivation: whey-based media, vegetable and fruit extracts, plant protein hydrolysates, and agro-industrial by-products [11]. However, none of the evaluated media was identified as a viable medium for the cultivation of LAB or as a low-cost alternative.

Sweet potatoes are a widely cultivated crop in the United States, with primary production concentrated in the southern states [12]. North Carolina in particular has been the leading producer of sweet potatoes since 1971, consistently accounting for a significant portion of the country's total production [13]. According to [14], North Carolina alone produced 61 percent of all sweet potatoes grown in the United States in 2019. Sweet potato is a root tuber that is rich in carbohydrates and minerals. On a dry basis, sweet potato contains 80–90% carbohydrate, of which 60–70% is starch. Other useful components present in sweet potatoes include proteins, vitamins, carotenoids, polyphenols, and minerals [15]. A previous study [8] demonstrated promising results using sweet potato as a suitable medium for LAB cultivation. However, in that study, an expensive, animal nitrogen source was utilized, similar to the one used in the MRS medium. Moreover, the formation of the sweet potato medium resulted in a liquid which created additional challenges related to handling and storage. [16] investigated the growth and viability of LAB cultivated with alternative nitrogen sources. That study demonstrated that the nitrogen sources (X-Seed Nucleo Max, and X-Seed KAT provided by Ohly GmBH, Germany) could efficiently support the growth and viability of LAB and be used for the fermentation of LAB.

Using sweet potatoes as a basic component in the development of a medium for the cultivation of LAB is an interesting approach to reducing the cost of the medium. Given the nutritional profile of sweet potatoes, including their high carbohydrate content, vitamins, minerals, and other beneficial compounds, they could provide a suitable foundation for a LAB growth medium. The objective of the present study was thus to investigate the use of sweet potato as a component in developing a medium for the cultivation of Lactobacillus.sp bacteria.

3.1 preparing Sweet Potato Powder.

The plant materials utilized in this study adhered to local or national guidelines.

Sweet potatoes (SP) were purchased from a local grocery store in Greensboro, NC. The SP was washed properly to remove the soil residue, or any chemicals that came from using fertilizers, and baked in a conventional oven at 200 ℃ for 30 min. The baked SP was then soaked in deionized distilled water (DI) at ratio 2:1 overnight at 4 ℃. then blended with a standard kitchen blender. This solution was centrifuged at 7000 rpm for 15 min (Thermo Scientific Sorvall ST 16R), and the supernatant was filtered using a cheesecloth. The sweet potato extract was collected and freeze dried (Harvest Right, HRFDL) overnight to form sweet potato powder (Fig. 1). The sweet potato powder was stored in a jar with desiccant bag to absorb the moisture at 4 ℃ for further used(Fig. 2).

3.2 Sources and preparation of Lactobacillus.sp bacterial strains for inoculation.

A total of seven bacteria strains were used in this study (Table 1). Three of Lactobacillus delbrueckii subsp. bulgaricus strains were isolated from commercial yogurt products available in the US market. Three Limosilactobacillus reuteri, and one Lactobacillus rhamnosus GG. All strains were obtained from the − 80°C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&T State University. All strains were activated in De Man–Rogosa–Sharpe (MRS) broth (Neogen Corporation, Lansing, MI, USA) by following the quality control procedure [17]. The Lactobacillus.sp strains were grown in MRS broth medium till reaching an optical density (OD₆₁₀) between 0.7 and 0.9 then individually centrifuged at 5000 rpm for 20 minutes at 4 ℃. The resulting supernatant was decanted, and cells were washed with 0.1M phosphate-buffered saline (PBS) solution and kept at 4 ℃ for further use.

Table 1

*Lactobacillus.sp* strains used in this study.
Bacteria Species	Strain Code	Sample	Original Source
Lactobacillus delbrueckii subsp. bulgaricus	B001	Yogurt	Spain
	B003	Yogurt	USA
	B006	Yogurt	USA
Limosilactobacillus reuteri,	ATCC5730	Pure Industrial Strain	ATCC
	MF14-C	Pure Industrial Strain	BIOGAIA Probiotics
	SD2112	Pure Industrial Strain	Humen milk
Lactobacillus rhamnosus GG	B 103	Pure Industrial Strain	Humen milk

3.3 The preliminary examination of sweet potato medium.

The sweet potato medium (SPM) was prepared by dissolving 100 g of sweet potato powder in 1 liter of DI water. The SPM was then autoclaved for 15 minutes at 120 ℃. The SPM was cooled down to room temperature and kept in the fridge for further use. To examine the growth of Lactobacillus.sp strains in SPM, a100µl of active bacterial strains from the third-fold dilution was inoculated in triplicate into SPM (10 ml), and MRS medium (10 ml) as a control. All samples were anaerobically incubated at 42 ℃ for L. bulgaricus strains and at 37 ℃ for L.reuteri, and L. rhamnosus strains for 12 h.

3.4 Culture growth in MRS, and SPM medium supplemented with different nitrogen sources.

Three yeast samples (X-Seed Nucleo Max, and X-Seed KAT) provided by Ohly GmBH, Germany, and (NuCel 780 MG) provided by Procelys, Biospringer, France were used and tested during this study. Table 2 highlights the total percentage of protein and nitrogen in X-Seed Nucleo Max, X-Seed KAT, and NuCel 780 MG respectively. After studying the growth behavior of all 7 strains, the strain was grown in MRS broth medium till reaching an OD₆₁₀ between 0.7 and 0.9 then individually centrifuged at 5000 rpm for 20 minutes at 4 ℃. The resulting supernatant was decanted, and cells were washed with 0.1M PBS. Washed cells were then ten-fold serially diluted in PBS. From the third-fold dilution, 100µl was inoculated in triplicate into each batch of test tubes (10 ml) as follows; MRS broth (control), SPM1 with nitrogen source (0.2% X-Seed Nucleo max), SPM2 with nitrogen source (0.2% X-Seed Nucleo max, 0.3% X-Seed KAT, and 0.3% NuCel 780 MG), and SPM3 with nitrogen source (0.2% X-Seed Nucleo max, 0.6% X-Seed KAT, and 0,6% NuCel 780 MG,) (Table 3). All test tubes with the different batches of nitrogen sources were anaerobically incubated at 42 ℃ for L. bulgaricus strains and 37 ℃ for L.reuteri, and L.rhamnosus strains for 12 h.

Table 2

Total protein (%) and nitrogen (%) for nitrogen source samples used in the study.
Yeast Samples	Protein (%)	Total Nitrogen (%)
X-Seed Nucleo Max	71	11.4
X-Seed KAT	75	12
NuCel 780 MG	62.5	10

Table 3

Components and nitrogen sources g/L used in growth media for *Lactobacillus.sp* bacteria.
Component	MRS	SPM1	SPM2	SPM3
Sweet potato powder	-	84.85	78.85	72.85
Dextrose	20	-	-	-
Peptone	10	-	-	-
Meat extract	10	-	-	-
Yeast extract	5	-	-	-
X-Seed Nucleo Max	-	2	2	2
X-Seed KAT	-	-	3	6
NuCel 780 MG	-	-	3	6
Sodium acetate (CH₃COONa)	5	5	5	5
Disodium phosphate (Na₂HPO₄)	-	2	2	2
Potassium phosphate (K₂HPO₄)	2	2	2	2
Ammonium citrate (NH₄C₆H₅O₇)	2	2	2	2
Magnesium sulfate (MgSO₄.7H₂O)	0.2	0.1	0.1	0.1
Manganese sulfate (MnSO₄.5H₂O)	0.05	0.05	0.05	0.05
Tween 80	1.08	1	1	1
L-Cysteine	-	1	1	1
Total (g)	55.33	100	100	100

3.5 Bacterial Enumeration.

Bacterial growth of all strains was monitored by measuring the turbidity (optical density at 610 nm) at different times (0, 6, 9, and 12 h) using a Thermo Fisher Scientific Evolution 201 UV-visible spectrophotometer (Thermo Fisher Scientific, Inc. Waltham, MA, USA). The uncultured media were used as blanks during the spectrophotometric measurement. The initial bacterial populations were determined at (0h) and the final bacterial populations were determined at the end of the fermentation period (12 h), a 1 mL of sample was drawn from each inoculated sample and was serially diluted in 9 ml 0.1% peptone water. Appropriate dilutions were then surface plated (100 µl) onto duplicate MRS agar plates. Agar plates for L. bulgaricus were anaerobically incubated for 72 h at 42°C and agar plates for L.reuteri, and L.rhamnosus were anaerobically incubated for 72 h at 37°C. Plates with 25–250 colonies were counted. The bacterial populations were then expressed in log CFU/mL.

3.6 Determination of buffering capacity and titratable acidity.

The buffering capacity (BC) and titratable acidity (TA) were measured using automatic potentiometric titrator (HANNA, H1931). BC was determined by placing 10 mL of the medium into a 100 mL beaker then titrated with 0.1 N HCl until pH 2.0 reached. TA was determined by placing 10 ml of the medium into 100 ml beaker then titrated with 0.1 N NaOH until pH 8.0 reached.

3.7 Determination of pH values.

The pH readings were taken using a pH meter (Accumet AB 150, Fisher Scientific, Pittsburgh, PA) after calibration with standard pH buffers 4.0, 7.0, and 10. The pH values of each test tube sample were measured in duplicates by placing the pH prop into the sample at the time of fermentation (0, 6, 9, and 12 h). The pH electrode was completely rinsed with DI and wiped between each measurement.

4.1 Growth and cell density of Lactobacillus.sp strains in sweet potato media.

The growth of Lactobacillus.sp strains in SPMs was monitored by measuring the turbidity through the detection of light scatter in absorbance at 610 nm (OD_610nm) and bacterial population (log CFU/mL). Our results showed that the growth in SPM without any supplements was low for all Lactobacillus.sp strains at 12 h of incubation and reach an average of 0.21 ± 0.08 which is significantly (p > 0.05) lower than the growth in MRS with an average of 1.35 ± 0.11 (see Fig. 3). For the bacterial population (log CFU/mL), the population of all Lactobacillus.sp strains was observed after 12 h of incubation. The average population showed a significant (p > 0.05) differences among MRS and SPM without any supplements (Fig. 4). The average population of Lactobacillus.sp strains in SPM was 4.16 log CFU/mL less than that in MRS which has an average of 8.04 log CFU/mL. Based on our results, sweet potato-based medium could be a potential as an effective substrate for Lactobacillus.sp cultivation.

SPMs were formed at different concentrations of plant- based nitrogen sources to lower the cost (Table 3). Figure 5 shows the growth of Lactobacillus.sp strains at 12 h of incubation at 42 ℃ for L.bulgaricus strains and 37 ℃ for L.reuteri, and L.rhamnosus GG strains. During the first 6 h of incubation, no visual growth was observed for all strains. The growth for all strains in SPM2 and SPM3 was similar or higher compared to that in MRS. SPM1 showed lower growth compared to MRS, especially in L.bulgaricus strains. The growth in MRS, SPM1, SPM2, and SPM3 measured by optical density OD₆₁₀ reached an average of 1.72 ± 0.13, 1.29 ± 0.1, 1.86 ± 0.13, and 1.93 ± 0.09 respectively at 12 h of incubation (Fig. 6). The growth of Lactobacillus.sp strains grown in SPM3 were significantly (p > 0.05) higher than that in MRS and SPM1, and there is no significant (p > 0.05) difference between SPM3 and SPM2, or between SPM2 and MRS.

Figure 7 shows the bacterial populations (log CFU/mL) for Lactobacillus.sp strains at 12 h of incubation. Only a slight difference among MRS, SPM1, SPM2, or SPM3 in the population of Lactobacillus.sp strains was observed except the two strains L.bulgaricus B001 and L.bulgaricus B006 in MRS and SPM1. Table 4 shows the average population (log CFU/mL) of lactobacillus.sp strains in MRS, SPM1, SPM2, and SPM3 which were 7.94 ± 0.11, 7.15 ± 0.13, 8.38 ± 0.11, and 8.57 ± 0.12 respectively. The average population of Lactobacillus.sp strains in SPM3 was significantly (p > 0.05) higher than that in MRS and SPM1. The results showed no significant (p > 0.05) differences among SPM1 and SPM2, or SPM2 and MRS. The findings regarding bacterial populations align with the optical density measurements (OD₆₁₀) demonstrating that SPM2 or SPM3 is suitable for the growth of the tested Lactobacillus.sp strains. Nitrogen plays a vital role as an essential component in fermentation media. It stimulates increased cell numbers throughout the fermentation process, leading to a greater capacity for producers to fulfill the minimum Colony-Forming Unit (CFU) requirements for their products [18].

Table 4

Averages of bacterial populations for *Lactobacillus.sp* strains in MRS, SPM1, SPM2, and SPM3 at 12 h of Incubation.
Medium	Average log CFU/mL
MRS	7.94 ± 0.11^b
SPM1	7.15 ± 0.13^c
SPM2	8.38 ± 0.11^ab
SPM3	8.57 ± 0.12^a

4.2 Titratable acidity (TA) of growth media MRS and SPMs.

The overall acid content of a food is measured by its titratable acidity. Food acids are typically organic acids, with the most prevalent types being citric, malic, lactic, tartaric, and acetic acids. These acids can occur naturally in food or may develop during fermentation processes. However, Food acidulation is frequently caused by inorganic acids, such as carbonic and phosphoric acids, which can have a significant effect. Foods' organic acids affect taste, color, and microbiological stability since organisms are inherently sensitive to pH [19]. Table 5 shows the TA values in MRS, SPM1, SPM2, and SPM3 at 6, 9, and 12 h of incubation with initial values of 1.63, 1.83, 2.17, and 2.23. respectively. There is a slight increase in TA values at 6 h of fermentation for all media. SPM2, and SPM3 showed significantly (p < 0.05) higher TA values compared to MRS, and SPM1 at 12 h of incubation. The growth of Lactobacillus.sp in SPM2, and SPM3 resulted in an average of 7.33 ± 0.08, and 7.76 ± 0.12 production of TA respectively. MRS, and SPM1 showed a low TA production with an average of 4.39 ± 0.11 4.68 ± 0.14 respectively (Fig. 8).

Table 5

Titratable acidity (TA) values for *Lactobacillus.sp* strains in MRS, SPM1, SPM2, and SPM3 at 6, 9, and 12 h of incubation measured by g/L.
Bacteria Strain	Incubation Time (h)
	6				9				12
	MRS	SPM1	SPM2	SPM3	MRS	SPM1	SPM2	SPM3	MRS	SPM1	SPM2	SPM3
L. rhamnosus GG	1.72± 0.12^d	2.24± 0.1^c	2.64± 0.08^b	3.05± 0.14^a	1.75± 0.06^c	2.38± 0.09^b	2.65± 0.13^ab	3.04± 0.11^a	2.3± 0.09^c	2.44± 0.15^bc	2.88± 0.07^b	3.37± 0.1^a
L. reuteri ATCC5730	1.65± 0.16^b	1.87± 0.13^b	2.57± 0.09^a	2.98± 0.11^a	2.51± 0.08^b	4.18± 0.11^a	4.25± 0.05^a	4.21± 0.13^a	5.71± 0.17^c	5.73± 0.09^c	10.35± 0.11^b	11.27± 0.08^a
L. reuteri MF14C	1.67± 0.09^c	1.95± 0.13^bc	2.35± 0.16^ab	2.77± 0.05^a	2.33± 0.1^c	4.55± 0.18^b	5.7± 0.08^a	5.0± 0.07^b	4.18± 0.06^c	6.0± 0.18^b	10.27± 0.09^a	10.1± 0.1^a
L.reuteri SD2112	1.74± 0.12^b	1.95± 0.05^b	2.32± 0.18^a	2.8± 0.14^a	3.0± 0.09^d	5.17± 0.13^c	8.2± 0.1^b	8.68± 0.1^a	4.5± 0.07^c	6.32± 0.18^b	10.0± 0.19^a	10.45± 0.08^a
L. bulgaricus B001	1.7± 0.13^d	2.01± 0.09^c	2.26± 0.07^b	2.66± 0.14^a	1.67± 0.08^d	2.02± 0.16^c	2.35± 0.09^b	2.76± 0.11^a	4.24± 0.07^b	3.3± 0.11^c	6.15± 0.16^a	6.3± 0.09^a
L. bulgaricus B003	1.7± 0.06^c	1.96± 0.12^bc	2.32± 0.08^ab	2.67± 0.13^a	1.93± 0.06^b	2.08± 0.18^b	3.14± 0.07^a	3.6± 0.11^a	8.34± 0.15^a	5.87± 0.08^c	6.03± 0.1bc	6.4± 0.07^b
L. bulgaricus B006	2.67± 0.1^ab	2.26± 0.07^b	2.62± 0.12^ab	2.97± 0.07^a	2.75± 0.09^ab	2.28± 0.13^b	2.73± 0.17^ab	3.01± 0.08^a	4.44± 0.1^c	3.1± 0.16^d	5.64± 0.09^b	6.45± 0.12^a
The initial TA are 1.63 MRS, 1.83 SPM1, 2.17 SPM2, and 2.23 SPM3 (Each value is the mean ± SD. The sample size is n = 3. Superscripts a–c denote significant (p < 0.05) differences between TA values of MRS, SPM1, SPM2, and SPM3 samples as determined by the Tukey-tests.)

4.3 Buffering capacity (BC) of growth media (MRS and supplemented SPMs).

SPMs were supplemented with different concentrations of plant-based nitrogen sources. The BC of SPM2 and SPM3 which contain 0.08% and 0.14% of nitrogen respectively was significantly (p < 0.05) higher than MRS and SPM1. In addition, there is no significant (p < 0.05) difference between MRS and SPM1. The highest value of BC was observed in SPM3 12.42 g/L, and the lowest value in SPM1 10.51g/L (Fig. 9). The addition of 0.08% or 0.14% of plant-based nitrogen source offered a more accurate assessment of the media's pH buffering capacity, which also demonstrated great promise by maintaining stable resistance to pH fluctuations over an extended period, while also effectively utilizing a significant portion of the 0.1N HCl solution. Figure 10 shows that SPM3 consumed 27.4 ml of 0.1N HCl to reach a pH point of 2, while MRS consumed 23.3 ml of 0.1N HCl to reach the same point of the pH. BC refers to the ability of a solution to resist changes in pH when an acid or base is added to it. It is a measure of the solution's ability to maintain its pH stability despite the addition of acidic or basic substances. Solutions with high BC can absorb or neutralize added acids or bases without significant changes in pH, whereas solutions with low BC are more prone to pH fluctuations. BC is influenced by factors such as the concentration and composition of the buffer system present in the solution [20].

4.5 pH Changes in the different growth media

In addition to the composition of the medium, various factors such as temperature, pH, oxygen levels, and water activity can influence the growth of Lactobacillus.sp. The ideal conditions for Lactobacillus.sp growth typically occur within pH levels ranging between 5.5–6.2. However, the Lactobacillus.sp genus is diverse, and bacteria within it can grow in pH levels varying between 4.5 and 6.5. Furthermore, certain strains are capable of growth even in environments with lower pH levels [21]. The initial pH for the tested media was between 6.1–6.5. After 6 h of fermentation the pH dropped slightly for all tested media and ranged between 5.8–6.4. The pH values for all tested strains after 12 h of fermentation were slightly different in all media. The highest pH value (5.84) was observed in SPM1 for L.rhamnosus GG, and the lowest pH value (4.11) was observed in SPM3 for L.reuteri, SD2112 (Table 6).

Table 6

profile of the pH values for MRS, SPM1, SPM2, and SPM3 due to the growth of tested bacteria strains.
Bacteria Strain	Incubation Time (h)
	6				9					12
	MRS	SPM1	SPM2	SPM3	MRS	SPM1	SPM2	SPM3	MRS		SPM1	SPM2	SPM3
L. rhamnosus GG	6.4± 0.08^a	6.0± 013^b	6.0± 0.06^b	5.92± 0.05^b	6.41± 07^a	6.0± 0.02^b	5.93± 0.06^b	5.81± 0.09^b	5.86± 0.09^a		5.84± 0.06^a	5.75± 0.1^a	5.63± 0.07^a
L. reuteri, ATCC5730	6.41± 0.11^a	5.92± 0.09^b	5.83± 0.13^b	5.81± 0.11^b	5.41± 0.08^a	5.33± 0.1^a	5.24± 0.08^a	5.21± 0.09^a	4.73± 0.12^a		4.64± 0.09^ab	4.31± 0.08^bc	4.28± 0.1^c
L. reuteri, MF14-C	6.31± 0.06^a	6.05± 0.02^b	5.92± 0.11^b	5.86± 0.08^b	5.91± 0.12^a	5.45± 0.08^b	5.42± 0.05^b	5.31± 0.09^b	4.71± 0.04^a		4.64± 0.12^a	4.45± 0.09^a	4.48± 0.06^a
L. reuteri, SD2112	6.33± 0.04^a	5.87± 0.1^b	5.84± 0.09^b	5.88± 0.07^b	5.82± 0.11^a	5.03± 0.09^b	4.83± 0.03^b	4.86± 0.08^b	4.82± 0.05^a		4.54± 0.1^a	4.17± 0.13^b	4.11± 0.08^b
L. bulgaricus, B001	6.42± 0.07^a	6.05± 0.12^ab	5.95± 0.09^b	5.84± 0.08^b	6.31± 0.14^a	6.0± 0.08^ab	5.82± 0.11^b	5.87± 0.09^b	5.11± 0.04^b		5.63± 0.11^a	4.65± 0.07^c	4.62± 0.13^c
L. bulgaricus, B003	6.41± 0.12^a	6.05± 0.04^ab	5.98± 0.08^ab	5.92± 0.1^b	5.35± 0.04^a	5.61± 0.13^a	4.72± 0.09^b	4.75± 0.11^b	4.32± 0.05^b		4.87± 0.12^a	4.64± 0.09^ab	4.62± 0.09^ab
L. bulgaricus, B006	6.39± 0.04^a	6.4± 0.13^a	6.31± 0.1^a	6.28± 0.09^a	6.31± 0.05^a	6.38± 0.03^a	6.3± 0.12^a	6.18± 0.09^a	4.85± 0.08^b		5.79± 0.06^a	4.88± 0.09^b	4.87± 0.12^b
(Each value is the mean ± SD. The sample size is n = 3. Superscripts a–c denote significant (p < 0.05) differences between pH values of MRS, SPM1, SPM2, and SPM3 samples as determined by the Tukey-tests.)

While the MRS medium is widely used for the cultivation of LAB and has many advantages, the popular medium also some significant drawbacks. For example, the preparation of MRS medium involves multiple ingredients and can be relatively complex compared to some other growth media. This complexity can increase the cost and time required for media preparation. Moreover, the formulations of MRS medium contain animal-derived ingredients such as beef extract and peptones, which may not be suitable for certain applications, particularly in industries focused on vegetarian or vegan products. Additionally, there are concerns related to allergens and cultural sensitivities associated with the use of animal-derived components. Our results revealed that SPM2 could be a suitable medium for the cultivation of Lactobacillus.sp. The utilization of sweet potato growth medium for cultivating Lactobacillus.sp presents an alternative to the traditional MRS growth medium. In addition, the present study highlights the remarkable buffering capacity of a sweet potato growth medium in comparison to the widely used MRS medium. The findings demonstrated that the sweet potato medium exhibited superior buffering properties, ensuring greater resilience against pH fluctuations during bacterial growth and fermentation processes. Not only did sweet potato medium offer comparable or superior growth outcomes for Lactobacillus.sp, it also presented obvious advantages in terms of cost-effectiveness. By utilizing the rich nutritional content of sweet potatoes, we can achieve robust bacterial growth by eliminating costly additives. Further study is needed in order to examine the SPM for cultivation of a wide range of Lactobacillus.sp including thermophilic and mesophilic LAB.

SPM: Sweet potato medium

LAB: Lactic acid bacteria

SP: Sweet potato

MRS: De Man–Rogosa–Sharpe

OD₆₁₀: Optical density

PBS: phosphate-buffered saline

BC: Buffering capacity

TA: Titratable acidity

Fundings:

This publication was made possible by grant numbers NC.X-267-5-12-170-1 and NC.X 359-5-24-170-1 from the National Institute of Food and Agriculture (NIFA) and the Department of Family and Consumer Sciences and the Agriculture Research Station at North Carolina Agriculture and Technical State University (Greensboro, NC, USA 27411). This work was also supported, in part, by 1890 Capacity Building Program grant no. (2020-38821-31113/project accession no. 021765).

Conflicts of interest:

Not applicable

Data availability:

All data generated or analyzed during this study are included in this published article.

Code availability:

Not applicable

Authors' contributions:

Abdulhakim Sharaf Eddin: wrote the draft of the manuscript, conducted the methodology, and interpreted the data. Salam A. Ibrahim: Designed and supervised the work, analyzed the data, and revised the manuscript.

Acknowledgements

Authors would also like to acknowledge the support of the Department of Family and Consumer Sciences and the Agricultural Research Station at North Carolina Agricultural and Technical State University (Greensboro, NC 27411, USA) and the entire research team in the Food Microbiology and Biotechnology Laboratory.

Lactic acid market size, share, growth, trends report, 2030. Lactic Acid Market Size, Share, Growth, Trends Report, 2030. (n.d.). https://www.grandviewresearch.com/industry-analysis/lactic-acid-and-poly-lactic-acid-market
Ravyts, F., Vuyst, L. D., & Leroy, F. (2012). Bacterial diversity and functionalities in food fermentations. Engineering in Life Sciences, 12(4), 356-367.DOI: https://doi.org/10.1002/elsc.201100119
Ayivi, R. D., & Ibrahim, S. A. (2022). Lactic acid bacteria: An essential probiotic and starter culture for the production of yoghurt. International Journal of Food Science & Technology, 57(11), 7008-7025.DOI: https://doi.org/10.1111/ijfs.16076
Fenster, K., Freeburg, B., Hollard, C., Wong, C., Rønhave Laursen, R., & Ouwehand, A. C. (2019). The production and delivery of probiotics: a review of a practical approach. Microorganisms 7: 83.DOI: https://doi.org/10.3390/microorganisms7030083
Saeed A, H., & Salam A, I. (2013). Current limitations and challenges with lactic acid bacteria: a review. Food and Nutrition Sciences, 2013.DOI:10.4236/fns.2013.411A010
Oleksy-Sobczak, M., & Klewicka, E. (2020). Optimization of media composition to maximize the yield of exopolysaccharides production by Lactobacillus rhamnosus strains. Probiotics and antimicrobial proteins, 12, 774-783.DOI: https://doi.org/10.1007/s12602-019-09581-2
Abbasiliasi, S., Tan, J. S., Ibrahim, T. A. T., Bashokouh, F., Ramakrishnan, N. R., Mustafa, S., & Ariff, A. B. (2017). Fermentation factors influencing the production of bacteriocins by lactic acid bacteria: a review. Rsc Advances, 7(47), 29395-29420.DOI: 10.1039/C6RA24579J
Hayek, S. A., Shahbazi, A., Awaisheh, S. S., Shah, N. P., & Ibrahim, S. A. (2013). Sweet potatoes as a basic component in developing a medium for the cultivation of lactobacilli. Bioscience, biotechnology, and biochemistry, 77(11), 2248-2254.DOI: https://doi.org/10.1271/bbb.130508
Ayad, A. A., Gad El-Rab, D. A., Ibrahim, S. A., & Williams, L. L. (2020). Nitrogen sources effect on Lactobacillus reuteri growth and performance cultivated in date palm (Phoenix dactylifera L.) by-products. Fermentation, 6(3), 64.DOI: https://doi.org/10.3390/fermentation6030064
Nochebuena-Pelcastre, X., Álvarez-Contreras, A. K., Hernández-Robles, M. F., Natividad-Bonifacio, I., Parada-Fabián, J. C., Quiñones-Ramirez, E. I., ... & Salinas, C. V. (2023). Development of a low pollution medium for the cultivation of lactic acid bacteria. Heliyon, 9(12).DOI: https://doi.org/10.1016/j.heliyon.2023.e22609
Hayek, S. A., Gyawali, R., Aljaloud, S. O., Krastanov, A., & Ibrahim, S. A. (2019). Cultivation media for lactic acid bacteria used in dairy products. Journal of Dairy Research, 86(4), 490-502.DOI: DOI: https://doi.org/10.1017/S002202991900075X
Smith, T. P., Stoddard, S., Shankle, M., & Schultheis, J. (2009). Sweetpotato production in the United States. The sweetpotato, 287-323.
Johnson, T., Wilson, N., Worosz, M. R., Fields, D., & Bond, J. K. (2015). Commodity highlight: Sweet potatoes. US Dept. Agr., Econ. Res. Serv. Pub. No. VGS-355-SA1.
2022 State Agriculture Overview. USDA/NASS 2022 State Agriculture Overview for North Carolina. (n.d.). https://www.nass.usda.gov/Quick_Stats/Ag_Overview/stateOverview.php?state=NORTH+CAROLINA
Akoetey, W., Britain, M. M., & Morawicki, R. O. (2017). Potential use of byproducts from cultivation and processing of sweet potatoes. Ciência Rural, 47, e20160610. DOI: https://doi.org/10.1590/0103-8478cr20160610
Ayivi, R. D., Ibrahim, S. A., Krastanov, A., Somani, A., & Siddiqui, S. A. (2022). The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii ssp. bulgaricus. Journal of Dairy Science, 105(10), 7986-7997. DOI: https://doi.org/10.3168/jds.2022-21971
Ayivi, R. D., Edwards, A., Carrington, D., Brock, A., Krastanov, A., Eddin, A. S., & Ibrahim, S. A. (2022). The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective. Vis. Exp, 184, e63314. DOI: 10.3791/63314
Yeboah, P. J., Ibrahim, S. A., & Krastonov, A. (2023). A review of fermentation and the nutritional requirements for effective growth media for lactic acid bacteria. Food Science and Applied Biotechnology, 6(2), 215-240. DOI: https://doi.org/10.30721/fsab2023.v6.i2.269
Nielsen, S. S. (2010). Food analysis fourth edition. Food Analysis, p. 227.
Silberberg, M. S., Amateis, P., Venkateswaran, R., & Chen, L. (2006). Chemistry: The molecular nature of matter and change(Vol. 4). New York: McGraw-Hill.
Śliżewska, K., & Chlebicz-Wójcik, A. (2020). Growth kinetics of probiotic Lactobacillus strains in the alternative, cost-efficient semi-solid fermentation medium. Biology, 9(12), 423. DOI: https://doi.org/10.3390/biology9120423

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Use of Sweet Potato as a Component in the Development of Laboratory Medium for the Cultivation of Lactobacillus.sp

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

3.1 preparing Sweet Potato Powder.

3.2 Sources and preparation of Lactobacillus.sp bacterial strains for inoculation.

3.3 The preliminary examination of sweet potato medium.

3.4 Culture growth in MRS, and SPM medium supplemented with different nitrogen sources.

3.5 Bacterial Enumeration.

3.6 Determination of buffering capacity and titratable acidity.

3.7 Determination of pH values.

Results and Discussion

4.1 Growth and cell density of Lactobacillus.sp strains in sweet potato media.

4.2 Titratable acidity (TA) of growth media MRS and SPMs.

4.3 Buffering capacity (BC) of growth media (MRS and supplemented SPMs).

4.5 pH Changes in the different growth media

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1