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Microbiology and Biotechnology Letters

Research Article(보문)

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Fermentation Microbiology (FM)  |  Applied Microbiology

Microbiol. Biotechnol. Lett. 2024; 52(2): 152-162

https://doi.org/10.48022/mbl.2312.12013

Received: January 12, 2024; Revised: March 14, 2024; Accepted: March 18, 2024

Modification of Substrate and Fermentation Process to Increase Mass and Customize Physical Properties of Lacticaseibacillus rhamnosus and Limosilactobacillus fermentum Exopolysaccharides in Kefir Grain

Dandy Yusuf1*, Raden Haryo Bimo Setiarto1, Andi Febrisiantosa2, Angga Maulana Firmansyah2, Taufik Kurniawan2, Ahmad Iskandar Setiyawan2, Rina Wahyuningsih2, Tri Ujilestari2, Satyaguna Rakhmatulloh3, and Heni Rizqiati4

1Research Centre for Applied Microbiology, National Research and Innovation Agency (BRIN), Jakarta-Bogor Street Km 46, Soekarno Science Centre, Cibinong 16911, West Java, Indonesia
2Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN), Yogya-Wonosari Street Km 31,5 Playen, Gunungkidul 55861, Yogyakarta, Indonesia
3Department of Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Fauna Street No. 3, Bulaksumur, Yogyakarta 55281, Indonesia
4Department of Agriculture, Faculty of Animal and Agricultural Sciences, Diponegoro University, Tembalang Campus, Semarang 50275, Indonesia

Correspondence to :
Dandy Yusuf,       mr.dandyyusuf@gmail.com

Abstract

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The microbial starter used to produce kefir beverages, kefir grain, contains a microbial exopolysaccharide called kefiran. Kefir grain consisting of water-insoluble polysaccharides, proteins, and fats, which can be applied as a multi-functional biopolymer. The mass of kefir grain can increase in the fermentation process of Kefir, but it is considered very slow. The purpose of this research is to study the impact of ammonium sulfate supplementation and yeast extract on reconstituted skim milk to increase the mass kefir grain and physical properties of kefiran. Results showed that the ammonium sulfate-supplemented substrate increased the mass of kefir grain by 547% in 14 days, with the condition that the substrate must be renewed every 2 days. Refreshing the substrate is considered one of the important factors. Supplementation on substrate did not appear to affect the viability of bacterial and yeast cells. Kefir grain produced from supplemented substrate also yields better thermal stability properties and has more functional groups than without supplementation. Two Lacticaseibacillus rhamnosus (RAL27 and RAL43) and one Limosilactobacillus fermentum (RAL29) were found to produce EPS. The three isolates also showed good skim milk fermentation ability after purification from kefir grain. The kefir grain produced in this study has the potential for wider application. This study also showed that kefir grain can be adjusted in quantity and quality through fermentation substrate engineering.

Keywords: Fermentation, kefir, kefiran, exopolysaccharide

Introduction

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Kefir is a fermented milk drink that involves several groups of microorganisms at once. The microorganisms involved include lactic acid bacteria, acetic acid bacteria, and yeast. These three groups of microorganisms live in a semi-solid matrix called kefir grain. Kefir grain is a starter that can be used repeatedly to produce kefir. In general, kefir grains increase their weight with subcultures in milk due to the increase in microorganism biomass and an increase in the amount of matrix that is composed of protein and microbial exopolysaccharide (EPS). Microbial EPS from kefir is then referred to as kefiran because it is different from other microbial EPS.

Kefir grain consists of water-insoluble polysaccharides, proteins, and fats. The physical characteristics of kefir grain are yellowish white, granule-shaped with a diameter of 2−15 mm, semi-solid texture, which is a gluco-galactan type EPS with glucose and galactose branch chains with a weight of about 10.000 kDa. Kefiran is a branching hexa- or hepta-saccharide repeating unit, according to its chemical components. It is made up of conventional penta-saccharide units to which one or two sugar residues are randomly connected. It has a backbone consisting of (1→6)-linked Glucose, (1→3)-linked Galactose, (1→4)-linked Galactose, (1→4)- linked Glucose and (1→2, 6)-linked Galactose, with branches attached to the O-2 of the Galactose residue and Glucose residues located at the ends of the structure [1]. The glycosidic bond diversity of kefiran is resistant to enzymatic degradation [2]. Lactic acid bacteria (LAB) are strongly suspected as a producer of EPS that form kefiran. Fujisawa et al. [3] have isolated Lactobacillus kefiranofaciens which is a capsular bacterium that produces kefiran. Other studies have also identified L. helveticus and L. pentosus as kefiran producers in Tibetan kefir grains [4, 5]. L. kefiranofaciens has also been identified to produce kefiran in single culture or in co-culture with Saccharomyces cerevisiae [1]. These studies show the potential of using single culture to produce Kefiran, but the researchers concluded that it is more effective to produce kefiran by capitalizing on the original kefir grain. Compared to other polysaccharides, Kefiran has superior functional properties, such as antibacterial, antifungal, and antitumor [6, 7]. In addition, there are potential benefits of Kefiran that utilize its structure or rheological properties, such as edible biofilm [8, 9], cryogel [10], biopolymer or biobased material [11, 12], and baker's yeast substituent [13, 14].

The mass of kefir grains, which contain kefiran, increases very slowly in the fermentation process, under general conditions only increasing by about 5−7% every 24−48 h [15]. However, studies have reported that kefir grain mass gain can be accelerated by adjusting the quality and quantity of nutrients in the substrate. Kefir grain mass can increase up to 500% after 20 days with substrate engineering and fermentation processes [16, 17]. Increasing kefir grain mass is thought to be possible with skim milk enriched with minerals and/or vitamins such as yeast extract and urea [15]. Another study by Harta et al. [17] reported that kefir grain mass increased rapidly in substrate containing carbon sources: fructose, glucose, or sucrose mixed with nitrogen sources: ammonium sulfate, potassium phosphate, or yeast extract. Another study reported that the production of kefir grain can be done by indigenous lactic acid bacteria culture of kefir grain, with adjustment of fermentation temperature, agitation level, and addition of carbon source, nitrogen source, vitamins, and minerals [18]. In more detail, to optimize kefir grains biomass or kefiran production, organic skim milk can be used, in addition to mineral sources and vitamins in sufficient quantities [19]. It can be assumed that to increase the production of kefir grain and kefir can be improved by optimizing the fermentation environment conditions and adjusting the quantity and/or quality of carbon and nitrogen sources. Food-grade carbon and nitrogen sources can be obtained from a variety of ingredients, including skim milk powder, ammonium sulphate ((NH4)2SO4), and yeast extract [16, 17].

After modifying the medium with the aim of increasing kefir grain production, it is necessary to analyze the effect on the characteristics of the Kefiran. This is increasingly important to determine the direction of proper utilization or application. The results of the study explained that generally EPS of lactic acid bacteria are used mainly in fermented milk technology may consist of glucose, fructose, galactose, rhamnose, xylose, mannose [20] in different compositions. Monosaccharides and the presence of various functional groups in kefiran can be identified by FT-IR through the observation of the presence of hydroxyl groups [6, 21, 22]. In addition, characterization of kefiran can also be carried out on polymer kefiran through thermal analysis techniques. One method that can be used is differential scanning calorimetry. Through this analysis, an explanation will be obtained that shows the characterization of the thermal properties and transitions of a polymer [22]. Kefiran also shows a homogeneous morphology with porous and a sponge-like structure when viewed with an electron microscope, which explains the high-water holding capacity [21]. The different characteristics of the kefiran set it apart from other common materials because of its unique microstructure [21]. Due to the above explanations, the purpose of this research is to study the impact of ammonium sulfate supplementation and yeast extract on reconstituted skim milk to increase the mass kefir grain and physical properties of kefiran.

Materials and Methods

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Preparation of kefiran

Kefir grains were obtained from a home industry in Bandung, Indonesia. Kefir grain in fresh condition or just separated from milk media before delivery, put in a cool box with a temperature of 4−10℃ and stored in the refrigerator before analysis. Kefir grains were activated by adding 50 g of kefir grain to 500 ml pasteurized reconstitute skim milk (15%) in Erlenmeyer flask and incubated at 25 ± 2℃ for 24 h. The grains were separated with a sterile 710 μm aperture sieve, reinoculated into fresh pasteurized reconstitute skim milk, and reincubated again at 25℃ ± 2℃ for 24 h. After two cycles of kefir grain activation process, the grains were considered ready for experiment.

Fermentation process to increase kefiran mass

Three separate duplicate trials were carried out using sterile 1 L Erlenmeyer flask prepared with 1 liter of either one of the following substrate types: skim milk (S); skim milk + yeast extract (SYE); skim milk + ammonium sulfate (SAS); and skim milk + yeast extract and ammonium sulfate (SYEAS). Skim milk were used at concentration 15% (w/v; NZMP, New Zealand) and both yeast extract (Pronadisa, Spain) and ammonium sulfate ((NH4)2SO4; Merck, Germany) were used at concentrations of 2% (w/v). Each customized substrate was inoculated with 15 g kefir grains then incubated at room temperature (25−30℃) for 14 days, which every 48 h, substrate was replaced.

Enumeration, screening, and isolation of microorganism

The enumeration, screening, and isolation of predominant lactic acid bacteria and yeast was referred to Yusuf et al. [25]. Approximately 25 g of kefir grain were homogenized in 225 ml phosphate buffer solution (pH 7.0; Himedia, India) for 15 min in a Stomacher (Bag Mixer, Interscience, France). The lactic acid bacterial and yeast contents were determined on MRS agar (Himedia, India) with 0.02% nystatin as anti-fungal and potato dextrose agar (Himedia) with 0.02% chloramphenicol as anti-bacteria, respectively. Approximately 100 μl solution from the 10-6 to 10-8 dilution was then spread onto agar medium in sterile plates, then incubated at 37℃ for 24−48 h. After the incubation period, the plates containing between 30 and 300 colonies were selected for enumeration. Specifically for LAB colonies, one colony was selected and then streaked on a new plate to obtain a pure colony.

Isolation and dry weight analysis

The method was referred to Schoevers et al. [16] with some adjustments. Kefir grains were separated with a 710 μm sieve, washed with sterile water, and carefully drained on tissue paper. Kefir grains were then weighed with an analytical balance. Furthermore, kefir grains were dried in a drying oven at 60−80℃ for 4−6 h or until a constant weight was obtained. Then, the dried kefir grains were analyzed for their physical properties. Moisture content and solids content were analyzed by the formula:

%Moisture = ((Mass initial − Mass dried) ÷ Mass initial) × 100

%Total solids = (100 − %Moisture)

Field emission scanning electron microscopy (FE-SEM)

FE-SEM experiments refers to Martins et al. [24] with some adjustments. The morphologies of the samples were characterized using FE-SEM Thermo Scientific Quattro S completed with EDS Detector. The dried kefir grains were powdered manually using a mortar and pestle. The powdered sample was placed directly on a stub that had been coated with carbon conductive tape. Next, the sample was coated with a thin film of gold. Sample is ready to be characterized.

Fourier transform infrared spectroscopy (FTIR)

FTIR experiments refers to Martins et al. [24] with some adjustments. The chemical structure of the materials obtained was characterized by FTIR in a Perkin- Elmer spectrometer, using universal attenuated total reflectance mode in the range of 4000−400/cm. The result obtained is the average of 16 scans. Resolution 4/cm and temperature 25℃.

Differential scanning calorimetry (DSC)

DSC experiments refers to Radhouani et al. [22] with some adjustments. DSC were conducted using ASTM F 2625-10 equipment, under a nitrogen atmosphere. About 5 mg of dried kefir grains were prepared and packed in aluminum pans. It was compared to an empty aluminum pan. The samples were heated in two stages at a constant heating rate of 20℃/min from 0℃ up to 250℃, then were left at this temperature during 2 min and cooled at 20℃/min to the initial temperature. Nitrogen atmosphere 40 ml/min.

Purification and physicochemical identification of isolates

Purification and physicochemical identification of isolates refers to the research of Yusuf et al. [25]. After 24− 48 h of incubation, single LAB colonies were then selected and streaked on fresh MRS agar by four-quadrant streaking. Scratching was repeated until a pure isolate was obtained based on the uniformity of cell shape observed under a microscope. After the pure isolate was obtained, Gram staining test and catalase test were conducted. LAB pure isolates were preserved in MRS broth mixed with 20% glycerol, then stored at -20℃.

Microbial exopolysaccharide production test

The method is based on Oleksy-Sobczak and Klewicka [26] with some adjustments. The isolate, which produced EPS, were tested on liquid EPS selection medium (ESM), namely MRS agar containing 20 g/l sucrose. The medium was streaked with LAB isolates and incubated at 30℃ for 2 to 3 days. After incubation, slimy colonies were determined.

Gene identification of isolates

The DNA extraction process was performed using GF- 1 Bacterial DNA Extraction Kit (Vivantis Technologies Sdn Bhd, Malaysia). DNA purity and concentration analysis was performed using Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). PCR amplification was performed in 20 μl volumes containing 10 μl of Promega Go Taq Green Master Mix 1x (M7122, Promega Corporation, USA), which contains Taq DNA polymerase, dNTPs, MgCl2, and reaction buffers; 1 μl of each primer 27F (5-AGAGTTTGATCCTGGCTCAG-3') and 1495R (5'-CTACGGCTACCTTGTTACGA-3') at a concentration of 0.5 μM each; 1 μl of DNA template (>150 ng); and 7 μl of nuclease free water (NFW; P1193, Promega Corporation). The amplification process was performed using an Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, USA) with the following conditions: 1 cycle at 95℃ for 5 min; 35 cycles at 95℃ for 1 min, 58℃ for 2 min, and 72℃ for 2 min; and 1 cycle at 72℃ for 10 min [27]. The PCR products obtained were then separated by gel electrophoresis (90V, 45 min) using 2% agarose gel in 1x TAE buffer, followed by staining using ethidium bromide. The PCR results were then sequenced using the Sanger method and then the results were analyzed by comparing the sequences stored in the NCBI GenBank database.

Characterization of isolates in reconstituted skim milk fermentation

The method is based on Yusuf et al. [25] with some adjustments. Each LAB isolate obtained was inoculated at 2% (v/v) into sterile reconstituted skim milk (12%; w/v), then incubated at 37℃ for 48 h. Observations were made on pH value (pH meter; Eutech pH 700) and conventional observations on whey amount, coagulant texture, and aroma. Visual and aroma identification was conducted by untrained individuals who were familiar with and could distinguish the typical sour aroma of fermented milk, alcohol aroma, and fruit aroma.

Total acid analysis was performed using the acid-base titration principle (AOAC 1995). A total of 10 ml of sample in an erlemeyer was added with three drops of 1% phenolphtalein indicator. The sample was then titrated with 0.1 N NaOH. The titration was stopped if the color turned pink. Percent total acid is calculated by the formula: Total lactic acid (%) = (NaOH volume × NaOH normality × 90 × 100) ÷ (sample volume × 1000).

Results

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Increased mass of kefir grain

The observation showed that the mass of kefiran increased significantly with the supplementation of 2% (w/v) ammonium sulfate and yeast extract in reconstituted skim milk (Fig. 1). Kefiran mass increased by 5−13 g after 2 days of incubation. The highest increase was in SYEAS substrate which increased by 13 g and SAS which increased by 8 g. After 6 days or 3 substrate changes, the significant effect of ammonium sulfate began to be seen, which increased the mass of Kefiran by an average of 10 g per 2 days.

Figure 1.Kefiran increased with the supplementation of yeast extract and ammonium sulfate in reconstituted skim milk. Different superscript letters are significantly different (p < 0.05, n = 3) by Duncan’s multiple range test.

Overall, in 14 days of incubation and 7 times substrate changes, the weight of Kefiran on substrate supplemented with AS increased by 547%, not significantly different from substrate supplemented with a combination of AS and YE, which increased by 540%. Whereas in the YE-supplemented substrate, the results showed an increase in kefiran mass of 140% and this was not significantly different from the un-supplemented reconstituted skim milk substrate, which was 153%. With these results, it can be seen that AS supplementation is essential, for the purpose of increasing Kefiran mass. Periodic substrate changes every 24−48 h are thought to be the key to accelerating kefir grain production. Substrate replacement also causes the fulfillment of kefir grain microbial nutrition. This result is in line with research reported by Schoevers and Britz [16] and Harta et al. [17].

In line with the above results, previous studies have reported that the production of kefiran can be done by modifying the fermentation substrate [16−18]. The addition of SA and YE is actually an effort to modify the substrate to affect the availability of organic and inorganic nitrogen sources. Wang and Bi [28] reported that the yield of kefiran in the MRSL was increased by 1.64 g/l and 0.69 g/l when YE and AS were added, respectively. Meanwhile, Zajsek et al. [18] also showed an increase in yield of kefiran of about 3.69 g/l but with the addition of ammonium nitrate. This study also confirmed that the production of the exopolysaccharide kefiran from the kefir grains can be dramatically enhanced by means of controlling the culture conditions, and modifying the milk medium's composition.

Moisture content of kefir grain in SYE, SAS, and SYEAS media is in the range of 78−83%. While the solid content is around 17−27%. While kefir grain cultured on S media, which can be referred to as normal grain, the moisture content is 85.5% and solid content 14.5%. The moisture content value of normal kefir grains has been published by Schoevers et al. [16] with a value of 82.6− 83.5%. The results in this study indicate the effect of media modification carried out to reduce the value of moisture content and increase solid content, as also reported by Wang and Bi [28]. Agitation treatment is also thought to affect the value of both [16].

Microorganism composition of kefiran

The results showed that substrate supplementation did not affect the growth of LAB and yeast groups. As shown in Table 1, the number of LAB and yeast colonies is relatively the same in each substrate tested. The majority of bacteria and yeast groups are thought to utilize SAS and SYE as a source of nutrients and produce certain metabolites, such as gluco-galactan which forms Kefiran. The difference in the weight of Kefiran produced should be expected to be influenced by the presence of species that have the ability to produce EPS or gluco-galactans. These species are thought to live in the kefiran tested, but the ability to grow may differ in a medium. For this reason, further research is needed to isolate and identify EPS-producing species. This is reinforced by the results of other studies that report L. pentosus, L. helveticus, and L. kefiranofaciens species isolated from Kefiran [4, 5] were identified to produce EPS. Meanwhile, from the yeast group, it is also suspected that there are species that have the ability to produce EPS. Some yeast genera that can produce EPS and are detected living in Kefiran include: Candida, Pichia, and Kluyveromyces [29, 30]. It seems that from the dominant LAB and yeast in Kefiran, isolates with the potential to produce EPS can be obtained.

Table 1 . Total colonies of lactic acid bacteria and yeasts on different substrate.

SubstrateaTotal colony (CFU/ml)
Lactic acid bacteriaYeasts
S2.12 × 1081.04 × 107
SYE0.43 × 1082.07 × 107
SAS1.65 × 1081.90 × 107
SYEAS1.15 × 1082.48 × 107

aS = Skim; SYE = Skim and yeast extract; SAS = Skim and ammonium sulphate; SYEAS = Skim, yeast extract, and ammonium sulphate.

Field emission scanning electron microscopy (FE-SEM)

Fig. 2 shows the characteristics of kefiran from substrate added with yeast extract and ammonium sulphate, which is covered by many microorganisms in the form of bacillus and coccus. The surface of kefiran has many gaps and fibers, which is characteristic of carbohydrate matrix. It can also be seen that the microbial cells that coat the surface of kefir are enveloped by a layer that is thought to be a biofilm. This is a defense mechanism of bacterial and yeast microorganisms. Bacterial and yeast cells also did not appear to be lysed, presumably because the heat exposure was carried out at low temperatures and lasted briefly. Kefiran from substrate added with yeast extract and ammonium sulphate appeared to contain more elements (Fig. 2G and 2H). This supports the notion that Kefiran microorganisms digest micro elements from yeast extract and ammonium sulphate to support their life. The figure also shows that the molecular structure of the Kefiran produced is different and is influenced by the nutrients available in the substrate.

Figure 2.FESEM images of dry kefiran from the skim and ammonium sulphate (A-D), skim + yeast extract and ammonium sulphate (E-H) substrate in magnitude 1,500-4,000x.

Fourier transform infrared spectroscopy (FTIR)

FTIR analysis was performed to characterize and identify the fundamental groups present in Kefiran. The results obtained in Fig. 3 show that there are bands at 3279/cm and 1629/cm corresponding to O-H hydroxyl groups in the constituent sugar residues. It is critical to emphasize that the reactive functional groups in polysaccharides allow them to be easily modified, which is critical for medical applications. Furthermore, hydrogels may be made fast and easily, making them ideal candidates for tissue engineering and regenerative medicine. The CH2 and OH groups are connected with the band around 1400/cm. The region 1100−1150/cm has demonstrated high absorption, which is typical of C-O-C stretching and alcohol groups in carbohydrates. The presence of bands at 900/cm shows configuration as well as glucose and galactose vibrational modes. These bands confirm that the compounds obtained are polysaccharides. The use of polysaccharides in tissue engineering is highly dependent on their physical behavior in addition to their chemical features [21, 31]. The graph's regions allow for the identification of more specific functional categories. Region 1 (3500−3000/cm) contains the band linked with the saccharidés hydroxyl group. Bands associated with symmetric and asymmetric extension of C-H bonds in methyl (CH3) and methylene (CH2) groups can be seen in area 2 (3000−2800). In area 3, O-H bond bending linked with water molecules is detected (2200−1500). Carbohydrate fingerprint region 4 (1000), related with ring elongation and side groups with C-O-C, C-OH, and C-H bonds, was detected. The results obtained in this study are in line with the results reported by Martins et al. [24].

Figure 3.Differences in kefiran functional groups in different substrate.

Differential scanning calorimetry (DSC)

DSC is a useful thermal analysis technique for determining thermal characteristics and polymer transitions (Fig. 4). The kefiran thermogram (endothermic heat flow) displays prominent endothermic peaks and differs between the four kefirans grown from different substrate. Kefiran produced with YE and AS were similar, and showed one endothermic peak at 111.2℃ and 107.5℃. The endothermic peaks of kefiran produced from skim and SYEAS were much higher and more than one peak, namely 128.4℃, 173.3℃, 184.2℃ in Skim and five peaks in the range 118.4℃−176.9℃ in SYEAS. This discrepancy can be due to their melting points, which is explained by the hydrophilic nature of Kefiran functional groups, the existence of these peaks may also indicate the presence of bound water. These results explain that kefiran with more thermal stability is produced on more complex substrates. It is suspected that the kefiran-producing microorganisms use the nutrient sources yeast extract and ammonium sulfate optimally to increase unique EPS or kefiran metabolites.

Figure 4.Differences in thermal properties and transitions of kefiran in different substrates.

Identification of exopolysaccharide-producing isolates

LAB screening and isolation procedures successfully obtained 10 pure isolates. All isolates showed bacil and coccus forms, as well as Gram positive and Catalase negative properties. Furthermore, the isolates were tested for EPS production ability on ESM media. The results showed that 3 isolates RAL27, RAL29, and RAL43 (Table 2) were able to form colonies and form a slime texture that was thought to be EPS [3]. The three LAB isolates suspected of producing EPS were then identified by the 16S rRNA gene. DNA extraction process has been done and obtained an average DNA concentration of 400−640 ng/μl, A260/A280 of 1.9, and A260/A230 of 0.5−0.9 (Table 3). The purity level of DNA can be determined by comparing the absorbance value of 260/280 nm. A good DNA purity value is between 1.8−2.0 [32]. This means that the concentration and purity of DNA obtained in this study have met the standards to proceed to the PCR process.

Table 2 . Identity of exopolysaccharide-producing LAB isolates.

Isolate nameCell morphologyGram stain resultCatalase productionEPS production
RAL27Rodpositivenegativepositive
RAL29Rodpositivenegativepositive
RAL43Rodpositivenegativepositive


Table 3 . Quality of DNA extract.

Isolates nameAveranges
ng/μlA260/A280A260/A230
RAL27555.151.940.66
RAL43641.651.930.53
RAL29406.251.940.9


The DNA component amplified by PCR was then observed by electrophoresis technique, the results are presented in Fig. 5. Through this process, it is known that the base length of the sample DNA is located at the same size of 1000 bp and the thickness of the DNA band indicates the quality of DNA purity is quite good. DNA bands that are thick and clustered (no smear) indicate a high concentration of DNA and are in intact condition [32].

Figure 5.Gel electrophoresis of DNA from RAL27 (A), RAL29 (B), and RAL43 (C) isolates.

Through the sequencing process, the 16S rRNA gene length of the three isolates is obtained more accurately, which is around 800 bp. Sequencing results that have been edited or contiguous then inputted to the BLAST website, showing isolates RAL27 and RAL43 have 100% similarity with Lacticaseibacillus rhamnosus and RAL29 has 99.89% similarity with Limosilactobacillus fermentum (Table 4). Base sequence similarity ≥ 94% can already be assumed that the isolates identified and homologous are the same species [33, 34].

Table 4 . 16S rRNA gen sequences.

NoIsolatesBasepairSpecies homolog in NCBI databasePercent identityAccession
1RAL27798Lacticaseibacillus rhamnosus100MT645513.1
2RAL43816Lacticaseibacillus rhamnosus100CP136120.1
3RAL29873Limosilactobacillus fermentum99.89OR520607.1


Characterization EPS production isolate in skim milk

The experimental results showed that the 3 LAB isolates produced fermented milk with a relatively similar profile (Table 5). Incubation for 24 h at 37℃ produced fermented milk with a pH range of 3.53−4.13. All three isolates produced fermented milk with compact coagulant and sour and refreshing aroma, and typical of fermentation. These results indicate that the three isolates that have been purified are thought to have consistent productivity to ferment skim milk as when in the SCOBY kefiran environment. In some isolates taken from a multispecies environment, the productivity will change due to loss of co-culture after isolation and purification [27]. The refreshing aroma produced is thought to come from ethanol and/or carbon dioxide (CO2). It is suspected that L. fermentum RAL29 belongs to the heterofermentative group of bacteria, which produce less lactate and less ATP, but produce several other end products. In heterolactic fermentation-type lactic acid bacteria, glucose can be broken down into lactic acid, ethanol, and CO2 [35]. However, this observation needs to be investigated further.

Table 5 . Characterization of skim milk fermented by EPS producing isolates.

Isolate nameTotal LAB (log CFU/ml)Quantity of whey*pHCoagulantAroma
L. rhamnosus RAL278++3.53CompactAcid
L. rhamnosus RAL438++3.93CompactAcid
L. fermentum RAL298+4.13CompactRefreshing acid

*10 ml of skim milk; ++ = 2.5 ml; + = < 2.5 ml.

Discussion

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The supplementation of ammonium sulfate and yeast extract was intended to affect the carbon and nitrogen ratio (C/N ratio). Nitrogen sources play a role in physiological processes as nitrogen is a component of proteins, nucleic acids and other important substances. The AS used changed the C/N ratio to 14.89. While the nitrogen source obtained from YE changed the C/N ratio to 28.44. The combination of the two changed the C/N ratio to 9.77. It is also suspected that other nutrients, such as sulfur, were affected by the use of AS. External factors are also thought to affect the results obtained. In this study, incubation was carried out at room temperature between 26−31℃. It is suspected that room temperature is unstable and may affect the overall activity of Kefiranproducing microbes. Different results may be obtained if incubators with controlled and stable temperatures are used.

The low and slow production of Kefiran has limited it’s in vivo and commercial applications. Therefore, strategies to increase the production of Kefiran have been studied. Based on the literature, Kefiran production was optimized by engineering culture conditions, engineering EPS biosynthesis genes, and metabolic engineering [29, 30]. Other researchers modified metabolic flux in recombinant strains by over-expressing NADH oxidase, allowing more carbon sources to be stored and thereby enhancing EPS synthesis. According to Ohba et al. [36], mild pulsed electric field (PEF) treatment generates electro-polarization in the cytoplasm, which influences the mobility of sugar nucleotides or the transport flexibility of repeating units via the membrane. Another reason that PEF therapy may promote EPS synthesis is because greater cytoplasmic membrane permeability makes more lactose available for EPS formation. Furthermore, stressful situations may induce strains to release more EPS in order to survive in such harsh environments [21]. Calcium and mild hydrogen peroxide, for example, can induce the overexpression of chaperonin, NADH peroxidase, and glyceraldehyde 3-phosphate dehydrogenase, enhancing energy storage and EPS synthesis. Cold stress changes the structure of L. sakei dextran while also boosting its production. Also, when the LAB strain is subjected to manganese stress, EPS synthesis may rise. This approach, however, may somewhat alter the structure of EPS, such as chain length or monosaccharide content. The use of skim milk powder and ammonium sulphate can be a strategy to save EPS production costs. In general, both can be used practically compared to cow's milk, which needs extra handling due to its perishable nature.

EPSs from microbes are quite diverse. Cellulose, emulsan, curdlan, gellan, levan, xanthan, and hyaluronic acid are some examples. Each is distinguished by the monomers and linkages that make up the EPS. The differences in EPS, in general, can be influenced by the type of species and the energy source or growth substrate [34]. The addition of yeast extract, ammonium sulfate or a combination of both, which was carried out in this study, is thought to produce uniqueness in Kefiran. However, further analysis is still needed to support the results of the FESEM analysis that has been carried out. L. fermentum RAL29, L. rhamnosus RAL27, and L. rhamnosus RAL43 can be utilized in single culture or co-culture for accelerated EPS production with supplementation of ammonium sufate and yeast extract. All three have the potential to produce unique and straint dependent EPS.

Acknowledgments

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All authors had equal contributions as the main contributors to this manuscript paper. The authors would like to thank LPDP Indonesia and BRIN Indonesia for fully supporting this research through the Program Research and Innovation for Advanced Indonesia (RIIM) 2023. Thanks to the facilities, scientific and technical support from the Bandung Advanced Characterization Laboratory, National Research and Innovation Agency.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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