In the present study, effect of xanthan gum was evaluated by using in vitro assay and skim milk model system on metabolic activity and survival of two probiotic strains, namely B. lactis and L. casei. In vitro assay was carried out identifying by pH, optical cell dencity (OD) and formation of postbiotics (lactic, acetic, propionic and butyric acids) in different basal media including glucose, inulin and xanthan gum as carbon source. The highest pH values were recorded for control (without carbon source) and media with xanthan gum, whereas the media with glucose and xanthan gum had the highest OD values. In comparison to strains, B. lactis had higher pH and lower OD values than L. casei. It was found that xanthan gum supported the formation of postbiotics as result of bacterial fermentation. In the skim milk model system, xanthan gum did not demonstrate a negative effect on probiotic viability, and the counts of both strains were above the required level for health benefits (8 Log cfu g-1) after 28-day storage. The use of xanthan gum in skim milk matrix positively affected technofunctional properties such as syneresis, color, textural parameters of samples. Consequently, xanthan gum is a good ingredient with multifunctional properties for industrial applications, and it also may be considered as an innovative approach to discover novel prebiotics.
Research Article
A novel approach to the use of xanthan gum: evaluation of probiotic promoter, postbiotic formation and techno-functional effect
https://doi.org/10.21203/rs.3.rs-1550126/v1
This work is licensed under a CC BY 4.0 License
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posted 15 Apr, 2022
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Xanthan gum
probiotic
postbiotic
milk
technofunctional effect
Xanthan gum is a microbial exo-polysaccharide obtained from various carbon sources by fermentation using Xanthomonas species such as X. campestris, X. pelargonii, X. phaseoli and X. malvacearum. The xanthan gum is formed by a β-D-glucose backbone with every second glucose unit linked through a trisaccharide consisting of D-mannose, and D-glucuronic acid and D-mannose. D-mannose units comprise acetic acid and pyruvic acid. This gum has anionic properties due to the carboxyl groups such as acetic acid and pyruvic acid on the side chains. It is identified as a GRAS food additive (E 415) by the European Union and the United States Food and Drug Administration (FDA). It has demonstrated tehnofunctional properties such as biodegradable, high viscosity and solubility at low concentrations in acidic, alkaline and salt solutions, pseudoplastic behaviour, excellent thermal stability, good mouthfeel, the ability to extend the shelf life, the potential antioxidant activity, and good freeze-thaw stability. Therefore, it has been used by the food industry for thickener, stabilizer, emulsifier, gel former, fat replacer, as well as an encapsulation agent. Moreover, since the backbone of the gum is a cellulosic β-(1,4)- D-glucan, it is resistant to variations in temperature, pH and shear stress and to degredation by various enzymes (GarcõÂa-Ochoa et al. 2000; Rather et al. 2015; Habibi and Khosravi-Darani 2017; Vega-Sagardía et al. 2018, Diantom et al. 2020).
When taking into account the complex heteropolysaccharide structure and enzymatically undegradable property of xanthan gum, it may show a promoter effect on the metabolism and growth of probiotic microbiota, and this gum might be considered as a candidate prebiotic as well as industrial importance. Even though probiotics are defined as live microorganisms that have beneficial effects on the health of the host when consumed in sufficient quantities, paraprobiotics, also termed inactiva, non-living microbial cells and ghost probiotics, have gained impotance in recent years due to their therapeutic properties (Hill et al. 2014; Omak and Yilmaz-Ersan 2022). Lactobacillus spp. and Bifidobacterium spp are the probiotic bacteria generally regarded as safe. Prebiotics are described as indigestible foods and/or food compounds that are selectively metabolized by beneficial microbiota mainly the Lactobacilli and Bifidobacteria in the large intestine and thus they positively affect the wellbeing and health of hosts (Gibson et al. 2017). The beneficial health effects attributed to bacterial cell mass, postbiotics (lactic acid, short chain fatty acids) and gases (CH4, CO2, H2,) formed by fermentation of prebiotics. Acetic, propionic and butyric acids with multiple health effects are the most common short chain fatty acids (SCFAs) via the saccharitic microbial fermentation. Recently, studies have focused on the production of novel ingredients with potential prebiotic activity from many various sources. In order to investigate the the prebiotic potential of a substrate or food, firstly in vitro model simulating fermentation processes is used, which provides a good system for small‐scale screening of novel substrates. The metabolism and growth of probiotic bacteria in this model are performed by measuring optical density, pH, acidity, counting viable cell and analyzing the quantity of postbiotics such as lactic acid and SCFAs. Secondly, prebiotic activity is confirmed by controlled animal and human clinical trials, mainly in vivo (de Souza Aquino et al. 2017; Khangwall and Shukla 2019).
However, the research into the probiotic promoter effect and fermentation characteristics of xanthan gum has not yet been implemented in detail. Previous studies mainly focused on the utilization of dairy products as a fat replacer (Nateghi et al. 2012; Murad et al. 2016; Murtaza et al. 2017), probiotic encapsulation (Chen et al. 2017; Nguyen et al. 2017; Tantratian et al. 2018) or a stabiliser (Mohsin et al. 2019). The objective of this research was to identify the fermentability of xanthan gum by some probiotic strains. The probiotic promoter effect was studied in both basal media and skimmed milk matrix with xanthan gum. The metabolism and survival of strains was tested by determining the pH, optical density (OD), quantity of postbiotics (lactic, acetic, propionic and butyric acids) in basal media. Furthermore, the present study investigated probiotic stimulating activity and technofunctional effect (e.g. stabilizer, fat replacer) of xanthan gum in the skimmed milk matrix during storage at 4°C for 28 days.
In vitro models system
Material
Bifidobacterium animalis subsp. lactis and Lactobacillus casei were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Both strains were activated at 37°C using an anaerobic condition (Anaerocult A, Merck, Germany). Xanthan gum was obtained from the As Food Co., (Istanbul, Turkiye).
Bacterial Basal Media Preparation and Inoculation
To test the effect of xanthan gum on the growth of B. lactis and L. casei, the basal medium carbohydrate-free, Tryptone Peptone Yeast Extract (TPY) for B. lactis and a De Man Rogosa ve Sharpe (MRS) broth for L. casei were prepared (Table 1).
The both medium were sterilized at 121oC for 15 min. Stock solutions of xanthan gum were prepared in distilled water and 0.45 µm-filter-sterilized (Millipore-Stericup-GP). In order to evaluate the effect of xanthan gum on metabolic activity and viability of B. lactis, based on the TPYcomposition, five different basal media were prepared firstly TPY without carbohydrates (negative control), secondly TPY+ glucose (0.50% w/v; Merck, Germany) and TPY + inulin (0.50% w/v Orafti® HSI, Belgium) as positive controls, and lastly TPY + 0.25% and 0.50% w/v xanthan gum (test substrates). Similarly, to investigate the effect of xanthan gum on L. casei, five different basal media were prepared using by MRS broth which was composed of glucose (0.5% w/v), inulin (0.5% w/v), xanthan gum (0.25 and 0.50% w/v) and without carbohydrate (negative control). 2% (v/v) of overnight probiotic strains, were inoculated into the TPY and MRS broth supplemented with glucose, inulin (0.5%) and xanthan gum (0.25 and 0.50%) as the sole carbon source.
Measurement of pH and optical cell density (OD)
The pH was determined at 12-hour intervals for up to 48 hours during fermentation using a pH-meter (pH 315i / SET; WTW, Germany). The OD was recorded at 600 nm absorbance with a spectrophotometer (Shimadzu UV 1800, Kyoto, Japan) at the 0, 12, 24, 24, 36 and 48th hour of fermentation. The sterile TPY and MRS broth without bacteria were used as blanks for the OD measurements.
Calculation of prebiotic activity score
In this assay, TPY and MRS broth supplemented with 0.50 % of xanthan gum and glucose were prepared for B. lactis and L. casei, respectively. A tryptic soy broth (TSB) supplemented with 0.50 % of xanthan gum and glucose were prepared for Escherichia coli as the representative enteric species. Afterwards, 1% of the inoculum of each strains were added to a TPY, MRS and TSB medium with 0.50% xanthan gum and glucose. Test cultures were incubated at 37°C for 24 hours anaerobically. Samples were taken at the 0 and 24th hour of fermentation, and the cell counts were determined by using TPY agar for B. lactis, MRS agar for L. casei and tryptic soy agar (TSA) for E. coli. Prebiotic activity scores (PAS) were calculated according to Huebner et al. (2008). 
Analytical methods for postbiotics
The postbiotics such as lactic acid and SCFAs were evaluated in the media with glucose (0.50%), inulin (0.50%), xanthan gum (0.25 and 0.50%) and without carbohydrate (control) after 48-hour fermentation. Standars for postbiotics were obtained from Merck, Germany. High Performance Liquid Chromatography (HPLC, Shimadzu Prominence, Japan) equipped in a Diode Array Detector (DAD, SPD-M20A) was used for the determination of lactic acid amount. The HPLC equipment consisted of the Shimadzu CTO-10ASvp Column. Analysis of short-cahin fatty acids was carried out on the Headspace Gas Chromatography (HS-GCMS, Agilent 7697A Headspace, USA). Samples were filtered with a 0.45 μm syringe filter and injected. Temperatures were set 35°C and 150°C for column, 200oC for dedector and 180oC for injector. Relative standard deviation values for the responses of the retention times obtained from standard solution mixtures containing different levels of acid were calculated for the repeatability of the method. Quantity of lactic acid and SCFAs was calculated as g L-1 (Fiori et al. 2018).
Skim milk model system
Culture activation
Freeze dried Bifidobacterium lactis (DANISCO, Sassenage, France) and Lactobacillus casei (Chr-Hansen, Istanbul, Turkiye) were used in milk model system. Reconstituted sterile non-fat milk (12 g 100 mL-1) was autoclaved at 121oC for 15 min for the culture activation. The strains were inoculated in sterile reconstituted skimmed milk and incubated anaerobically at 37±1°C (Anaerobic System Anaerogen, Oxoid, Basingstoke, UK).
Production of skim milk model system with xanthan gum
Skim milk powder was reconstituted in pure water at 10.70% (w/v). Four groups of milk model systems were manufactured by using probiotic strains and xanthan gum; BL (milk with B. lactis), BLG (milk with B. lactis and 0.25% xanthan gum), LC (milk with L. casei) and LCG (milk with L. casei and 0.25% xanthan gum). Reconstituted milk was divided into two experimental batches; one batch of which was supplemented with xanthan gum to final concentrations of 0.25% w/v. Each batch was subjected to pasteurization at 90°C for 10 minutes and subsequently cooled to 37°C. The samples were inoculated with the probiotic strains. The inoculum levels of each strain were determined to give a final concentration of approximately 8–9 log10 CFU mL-1 in milk and then incubated at 37°C until the final pH of 4.7 was achieved. The samples were cooled and stored for 28 days, which were then analyzed for probiotic viability, technofunctional properties (titratable acidity, syneresis, color and textural attributes) at days 1, 7, 14, 21 and 28 of cold storage (4°C.)
Enumeration of probiotic viability in milk model system
B. lactis and Lb. casei counts were enumerated by using Man, Rogosa and Sharpe agar (MRS; Merck, Germany) (Tharmaraj and Shah 2003). The plates were incubated at 37°C for 72 h anaerobically (AnaeroGen Gas Packs, Oxoid, Basingstoke, UK). Plates containing 30–300 colonies were counted and converted into log10 colony forming units (cfu g-1). Viability proportion index (VPI) of microorganisms was calculated according to the following formula:
Evaluation of technofunctional properties in milk model system
Total titratable acidity (TTA) was determined by the titration method and expressed as a percentage of lactic acid equivalents (AOAC 2000). In order to evaluate syneresis, 25 g sample was weighed and then filtered through Whatman paper at 4°C for 2 h. The syneresis was calculated as the weight of collected whey divided by the initial sample weight (Ozcan et al. 2020). Color parameters (L*, a* and b*) of samples were measured by using a Minolta Chromameter (Konica Minolta Co., Ltd., Osaka, Japan). L* parameter ranges from 0 (black) to 100 (white), a* shows from red (+a*) to green (−a*), and b* varies from yellow (+b*) to blue (−b*). Textural characteristics of samples were determined using a texture analyzer TA-XT Plus (Stable Micro System Ltd, Model TA-XT plus, UK) with the back extrusion method fitted with a 5 kg load cell. A 40-mm-diameter cylinder probe was used and penetrated the samples to 75% of their original depth. The speed of the probe was arranged at 0.1 mm/s during the pretest compression and relaxation of the samples. Firmness, consistency, cohesiveness and viscosity indexes were calculated using the Texture Expert Exceed software (v 2.55) extracted from the resulting force time curves (Patrignani et al. 2007).
Statistical analysis
The results obtained from the present study were statistically evaluated using analysis of variance (ANOVA) and Fisher’s least significant difference (LSDs) method (p<0.05 and p<0.01, Minitab 17, USA). Significant differences statistically were indicated using different letters. The hierarchical cluster analysis was performed following an unweighted pair group method with an arithmetic average based on a dissimilarity matrix. In order to analyze the relationship among tehnofunctional properties of skim milk matrices, the Pearson Correlation Coefficient (r) was calculated on individual data using the Statistical software (IBM SPSS Statistics, USA).
In vitro system
Two probiotic strains, B. lactis and L. casei, were selected for the fermentation studies. These species probably differ in their capability for growing on xanthan gum-derived components. Fig. 1 shows that the pH values of the medium decreased during fermentation. Throughout fermentation, average pH values for B. lactis varied between 5.01 and 6.55. pH values for B. lactis in the medium with inulin and 0.50% xanthan gum were found statistically similar at the 24-hour-fermentation, whereas these values showed a decrease after 24-h-fermentation. In the present study, it was found that the pH values identified for B. lactis were similar to the values revealed in the study of Polari et al. (2012) in which they used galactoglucomannan extracted from spruce (Picea abies) as a carbohydrate source. Average pH values for L. casei ranged from 5.09 to 6.47 and showed a significant decrease during fermentation. It was found that the pH values identified in the 0.25% and 0.50% xanthan gum medium fermented with L. casei were lower than the control sample that did not contain any carbohydrate source. It was found that control (without carbohydrate sources) had the highest pH value for L. casei at the end of fermentation. Fig. 1 shows that the OD values of the medium were correlated with increased B. lactis, whereas those of L. casei showed an increase during 24-h-fermentation. It was found that the B. lactis in the media containing 0.50 % xanthan gum had the highest cell density value after media with glucose, which may mean the bifidogenic potential of xanthan gum. B. lactis in the media with 0.25 % xanthan gum reached the lag phase after 24-h-fermentation, whereas B. lactis in the media containing 0.50 % xanthan gum reached the lag phase after 36-h-fermentation. It was found that the cell density values identified for B. lactis in the study were similar to the values found in the studies by García-Cayuela et al. (2014) and Usta and Yilmaz- Ersan (2017) who studied the growth of B. lactis in different substrates. Table 2 shows the pH and OD values after the five fermentation times (0, 12, 24, 36 and 48 hours) in anaerobic culture. The results are presented as the mean value of each fermentation time, regardless of the different carbon sources and probiotic strains. There were significant differences (P<0.01) within the level of pH and OD values for different carbon sources, strains and fermentation time. Significant interactions among parameters were detected (P<0.01). Regarding the carbon source, the highest pH values were recorded for control and media with xanthan gum. When compared with the probiotic strains, B. lactis had higher pH and lower OD values than L. casei. A significant decrease in the pH L. casei was matched by increases in OD values, which means that xanthan gum had better growth-promoting effect for L. casei.
PAS of xanthan gum and inulin against glucose for B. lactis and L. casei were investigated, and the species showing positive PAS were shown in Fig. 2. The prebiotic activity scores for both strains were found to be higher than zero, which refers the fact that growth of the strains is higher for xanthan gum compared to the glucose, as well as higher than the reference bacteria (E. coli). Furthermore, it was found that PAS for L. casei in media with xanthan gum was higher than that of the inulin, whereas for B. lactis was lower than inulin.
In this study, for both strains, lactic acid was the most abundant produced metabolite. Figure 2 illustrates the lactic acid concentrations of B. lactis and L. casei in media including different carbon sources after 48-h-fermentation. The quantity of lactic acid varied from 1.10 g L-1 of B. lactis in media with glucose to the 15.74 g L-1 of L. casei in media without carbohydrate (control), depending on the strain and type of subtrate. More lactic acid was produced by L. casei than B. lactis. There were significant differences in total SCFA values among the strains depending on the substrate type used after 48-h-fermentation (Fig 2). Acetic acid used as energetic substrate in muscle tissue is the most abundant SCFA produced by the human colonic microbiota (Fukuda et al., 2011; Usta-Gorgun and Yilmaz-Ersan 2020). The acetic acid concentrations of strains were shown in Figure 2. Its quantity ranged from 0.03 g L-1 of B. lactis in media with xanthan gum (0.25%) to 2.55 g L-1 of L. casei in media with glucose. Propionic acid formed as a result of colonic fermentation can be utilized in liver cells. Propionic acid values varied from 0.002 g L-1 of B. lactis in media with inulin and xanthan gum (0.25%) to 0.428 g L-1 of the same microorganism in media with glucose. Butyric acid is important for the prevention and treatment of colonic diseases (Pessione et al., 2015). A maximum butyric acid value (0.50 g L-1) of L. casei fermented in the control (without carbohydrate) was observed, while B. lactis fermented in media with inulin had a lowest butyric acid value (0.03 g L-1). Generally, higher butyric acid values were present in media with xanthan gum than inulin (positive control). The quantity of lactic and total SCFAs was presented as the mean value of the different carbon sources and probiotic strains (Table 3). There were significant differences (P<0.01) within the level of lactic acid and total SCFAs for different carbon sources and strains. Regarding the substrate type, the highest lactic acid was recorded in the media without carbohydrate (negative control) whereas glucose was the main contributor substrate for SCFAs. The tested acids increased significantly (P<0.01) during the 48 h fermentation as a result of the bacterial enzymes. As with the lactic acid values, a significant interaction was detected (P<0.01) between “carbon sources and strains”.
In hierarchical cluster analysis, the mathematical relationship among different carbon sources on the growth of B. lactis and L. casei is calculated using the results of attributes (pH, OD, lactic and SCFAs) analyzed throughout the present study (Fig. 4). In respect to the cluster analysis results for B. lactis, inulin classified as a prebiotic source and xanhan gum revealed high similarity. As a result of the clustering analysis for L. casei, whereas high similarity was found between glucose and inulin, xanthan gum (0.25 and 0.50%) was closer to positive controls than negative.
Skim milk model system with xanthan gum
The utilization of xanthan gum by B. lactis and L. casei in skim milk model system was studied during the 28-day-storage, and the results were shown in Table 4. It was found that the viability of B. lactis and L. casei was found to be influenced by the xanthan gum except for the 7th, 14th and 21st days (P<0.01). At the first day of storage, the log number of B. lactis was 9,30 log10 cfu/g for sample with xanthan gum and 9,44 log10 cfu/g for sample without gum. It should be noted here that B. lactis count presented a continuous increment up to 7 days of storage, and the highest B. lactis counts were observed after one week in both matrices. A steady decrease in B. lactis numbers was noticed at the end of storage where the final number reached 8.29 log10 cfu/g for sample without gum and 8.05 log10 cfu/g for sample with gum. L. casei counts were identified as 9.90 log10 cfu/g for sample without gum and 9.47 log10 cfu/g for the sample with gum at the first day of storage. L. casei counts were stable during storage and the maximum counts were observed at the end of storage. At the end of storage, the VPI values for both strains ranged between 0.87-1.02. It was found that there was a relationship between the acidity increase and viability of L. casei.
The titratable acidity values of samples displayed significant differences depending on skim milk matrix and storage time (P<0.01, Table 5). The titratable acidity of control and samples with gum varied from 0.67 to 1.29 % throughout the storage time. The acidity of samples except for BL sample showed a significant decrease during storage. It was determined that the syneresis defined as the separation of the aqueous phase from continuous phase or gel network was found to be significantly influenced by the sample type and storage time (P<0.01, Table 5). The highest syneresis was identified in sample fermented L. casei at the end of the storage, whereas the lowest value was obtained in sample with gum. It was noticeable that the BL sample exhibited lesser syneresis values than LC sample after the beginning of the storage.
Statistical analysis illustrated that the addition of xanthan gum and storage time had a significant (P<0.05) effect on the color parameters (Table 5). Generally, samples without gum (BL and LC) had lower L* values than others and L* values of these samples decreased during the first seven days of storage. The a* values, with all minus measurements, confirmed that the greentone was dominating over the red in all samples. The samples including gum (BLG and LCG) had higher a* values than others without gum, whereas b* values of these samples were the lowest compared to the others. The b* values, with all measurements above zero, confirmed that the yellow coloration was dominating over the blue in all samples. During storage time, generally the a* and b* values of all samples increased, except for a* values of LC sample.
It was revealed that there were significant differences (P<0.01) between the textural parameter values during storage (Figure 5). There were significant differences in the use of xanthan gum and storage time for firmness values (P<0.05). Generally, the highest firmness values throughout storage was detected in sample formulated with gum and L. casei (LCG). It was found that the consistency values were found to be statistically different among the samples during storage (P<0.01). The maximum levels of consistency values were observed at the beginning and end of storage for LCG sample. While the utilization of xanthan gum resulted in an increase in cohesiveness values of sample except for those fermented by L. casei, in matrix fermented by B. lactis resulted in a decrease. The cohesiveness values of all samples except for LC sample during storage showed an increase. Higher cohesivenees values were detected for BL sample at the beginning of storage, whereas BLG and LCG samples had higher values at the end of storage. There were significant differences in the sample type and storage time for viscosity values (P<0.01). During storage, the viscosity values of BL and LCG samples decreased (P<0.05) while those of BLG and LC samples showed statistically insignificant differences (P>0.05).
Pearson’s correlation coefficients (Table 6.) were used to understand the use of xanthan gum in skim milk among the technofunctional properties. It was found that a strong positive correlation existed between the probiotic cell couts and titratable acidity (r=0.998; P<0.01). Syneresis was significantly positively correlated with L* (r=0.950, P<0.05), a* (r=0.986, P<0.05), appearance (r=0.954, P<0.05) and structure (r=0.990, P<0.01). L* was significantly positively correlated with a* (r=0.973, P<0.05), b* (r=0.998, P<0.05).
In vitro system
A decrease of pH occurs as a result fermentation of various carbon sources by strains in the in vitro system during fermentation, and it reflects the accumulation of postbiotics such as lactic acid and SCFAs. Of the both species compared with media containing xanthan, L. casei could develop acidity by activating in the media containing xanthan gum and reached to the lowest pH during the 48 h-fermentation due to a higher capacity to produce acids. All of the in vitro fermentation systems for both strains showed a significant decrease in their pH values after 48 hours, which is most probably caused by the fermentation metabolites such as lactic acid and SCFAs. The decrease in pH during fermentation, preventing the growth of harmful microorganisms and increasing the survival of beneficial ones lead to positive results in the colonic ecosystem. It was found that due to the structural properties of xanthan gum, the pH values in the present study turned out to higher than the values identified by Yılmaz-Ersan et al. (2018) and Karlton-Senaye and Ibrahim (2013). When the OD results were examined, it was found that in the MRS broth containing 0.25 and 0.50% xanthan gum, the L. casei had higher cell density value than inulin as the positive control. L. casei can develop in media containing the xanthan gum as well as in inulin. The increase in the cell dencitis of L. casei may be related to the high amounts of β-D-glucose in xanthan gum. Alarifi et al. (2018) tested the efficacy of gum acacia and fructooligosaccharide in batch cultures inouculated with human faeces over 48 hours. They found that gum acacia caused an increase Bifidobacterium spp. and Lactobacillus spp. similar to those of fructooligosaccharide. In addition, it was revealed that the 1% concentration of substrates demonstrated more increased selectivity compared to the 2% concentration. As pH, a function of the total hydrogen ions (H+) forms from acid production in the media, and alkaline property of xanthan gum may react with H+ to neutralize acidity. The highest OD values were detected in the media with glucose and xanthan gum. This suggests that xanthan gum was used by strains as carbon source. The pH values decrased significantly, while OD values increased during the 48-hour fermentation as a result of the bacterial enzymes. García-Cayuela et al. (2014) found that the OD values of B. lactis BB-12, B. breve 26M2 and B. bifidum HDD541 with the different carbohydrates including lactulose, lactulosucrose, lactosucrose, glucose, kojibiose and 40-galactosyl-kojibiose used as carbon sources varied between 0 and 1.21 after 48-h-fermentation. The metabolic activity and growth of probiotics are affected by chemical structure, composition of monomer units, degrees of polymerization (DP) and water solubility of carbohydrate sources (Voragen 1998).
PAS measures the changes in the cell counts and/or cell density values of probiotics to those of opportunistic microorganism as quantitative score. The positive PAS obtained for testing different carbon sources indicates that the carbon sources are fermented by probiotic bacteria preferably better than glucose, and increases probiotics but not other undesirable bacteria such as Enterobacterium spp. (Omak and Yilmaz-Ersan 2022).
Postbiotics, the new probiotic-related term, are the secondary metabolites formed by probiotics, which provide beneficial health effect on the human. Postbiotics comprise short-chain fatty acids, organic acids (lactic acid, 3-phenyllactic acid), secreted and/or extracellular polysaccharides, carbohydrates, bacteriocins (bifidin and acidophilin), enzymes, peptides, vitamins, lipoteichoic acids, and peptidoglycan-derived muropeptides. Especially, lactic acid and SCFAs formed via the glycolytic pathway during in vitro and in vivo fermentation offer beneficial healh effects such as antitumorigenic, antiobesogenic, antiallergic, diminishing of intestinal infections and increasing mineral bioavailability (Omak and Yilmaz-Ersan 2022). The source and chemical structure of substrates, the microorganism population, and the gut transit time affect SCFAs formation which is an important parameter for evaluating the fermentation capacity of microorganisms used with substrates. There are no studies concerning the formation of these postbiotics in media with xanthan gum. It was evaluated that higher lactic acid and total SCFAs were produced by L. casei. Lactobacilli can use the glycolytic pathway and the phosphoketolase pathway under the heterofermenting conditions, whereas Bifidobacteria are unable to use of the usual glycolytic pathway or the hexose monophosphate shunt pathway due to a lack of aldolase and glucose-6-phosphate NADP+ oxidoreductase. Differences in the metabolic pathway between strains and subtrate types influenced the quantity of posbiotics. Taking into account the above-mentioned results, the media without carbohydrate had higher total SCFAs because the majority of acids might be consumed by strains in media with xanthan gum.
In hierarchical cluster analysis. the classification of from the highest to the lowest for both strains, is as follows: control > glucose > inulin > xanthan gum (0.25 and 0.50%). These results indicated that xanthan gum could show stimulating effect on the growth B. lactis and L. casei and their formation ability of acidity and SCFAs during the in vitro anaerobic fermentation.
Skim milk model system with xanthan gum
Although there is no universally accepted probiotic microorganism number for beneficial health effect, the recommended number ranges from 6 log cfu mL-1 to 8 log cfu g-1 (EFSA 2010; Codex Alimentarius 2011; Mousavi et al. 2019) in the fermented product. The results of this study indicated that high levels (7 log cfu g and/or mL-1) of both of strains could be maintained at the end of storage. Generally, it was determined that the highest VPI throughout the storage was determined for L. casei. However, the stimulation effect of gum on the L. casei counts may be attributed to its buffering capacity due to acid and enzymatic hydrolysis. It was found that xanthan gum was found to be less helpful in the viability and activity of probiotics in skimmed milk matrix, presumably due to its highly branched and inherently complex structure. However, there have been only few studies on the prebiotic potential of xanthan gum in food matrix. Ziaolhagh and Jalali (2017) investigated the effect of kakuti or wild thyme essence and xanthan gum on viability of B. lactis in doogh. They reported that by increasing the amounts of xanthan gum to 0.075%, B. lactis count increased as well, but increasing the gum further had no significant effect on microorganism number. Karlton-Senaye et al. (2015) stated that the viability of L. reuteri strains in milk drink samples containing gums remained stable during the first 2 weeks of storage and then decreased slightly, whereas viability of Lb. rhamnosus GG B103 and Lb. rhamnosus GG B101 remained stable during storage. Similar to our findings, zedo gum showed insignificant effect on the survivability of L. acidophillus and B. bifidum during 29 day-storage (Ghasempour et al. 2012). Bahrami et al. (2013) stated that the statistical analysis showed that xanthan gum, guar gum, and barley beta-glucan additions had no marked effect on L. bulgaricus, S. thermophilus and L. acidophilus counts of yogurt samples. El-Sayed et al. (2002) determined that the use of xanthan gum and mixes of guar gum, carboxymethyl cellulose and locust bean gum in yogurt and soy yogurt did not affect the counts of probiotic bacteria.
The samples fermented by Lb. casei had the highest acidity at the begining and end of the storage. Ziaolhagh and Jalali (2017) found that the acidity increased in the yogurt drink including kakuti or thyme essence and xanthan gum during storage and the samples containing xanthan gum had the highest acidity. El-Sayed et al. (2002) reported that the use of xanthan gum or its mixtures had an insignificant effect on the acidity values of yogurt or soy yogurt samples compared to the corresponding controls. The type and amount of the produced organic acids in the presence of gums on depending on probiotic strains specific resulted in the different acidity values in various studies (Ghasempour et al. 2012; Bahrami et al. 2013; Karlton-Senaye et al. 2015). Syneresis results may be explained by the fact that the increase of acidity in LC sample during storage altered the development of a more open structure of the sample network and higher syneresis values. Ghaderi-Ghahfarokhi et al. (2020) and Prasanna et al. (2013) reported that exopolysaccharides produced by Bifidobacterium species could hold free water, reduce the sineresis and create firmer fermented milk gels. The findings showed that the syneresis decreased during cold storage for samples including gum. The addition of gum to milk products can retard syneresis or improve yogurt texture due to the interaction between the gum and milk protein concentration (Ghaderi-Ghahfarokhi et al. 2020). Bahrami et al. (2013) reported that syneresis decreased with an increased xanthan gum concentration of up to 0.2%, but then syneresis of the yogurt samples increased.
The color is an important parameter which affects consumer acceptability and marketing value of a product and is directly influenced by the ingredients and starter culture used in the formulation (Topcuoglu and Yilmaz-Ersan 2020).
Firmness, the force necessary to attain a given deformation, is the higher value means a firmer sample. Mohsin et al. (2019) reported that maximum firmness was found in the camel milk date yoghurt including 0.5% and 0.75% biosynthesized xanthan gum. The higher consistency refers to a high viscous or thicker sample. Since cohesiveness is related to the strength of the internal bonds in fermented milk structure, the lower the cohesiveness the smoother the product texture. While the utilization of xanthan gum resulted in an increase in cohesiveness values of sample except for those fermented by L. casei, in matrix fermented by B. lactis resulted in a decrease. The higher values of index of viscosity refer to being more resistant to gradual deformation of the sample by shear stress, meaning thickness. A greater value in the index of viscosity were observed at the beginning of storage for LCG sample, whereas at the end of storage BL sample had the highest value. This may be attributed to the combined effect of polysaccharides produced by Bifiobacteria and low acidity activity of these strains. Ziaolhag and Jalali (2017) reported that the viscosity of probiotic yogurt drink increased as the concentration of xanthan gum increased from 0 to 0.15%. The interaction of the negatively charged xanthan gum with the positively charged surface of the casein micelles in milk contributes to the highly structured network formation, increasing sample viscosity and gel strength. (Nguyen et al. 2017). In this study, it was determined that the textural differences were found to be significant among the samples, depending on the probiotic type and xanthan gum, and it was observed that the textural parameters increased for matrix including xanthan gum and L. casei (LCG). Thus, further studies are required to characterize the hydrophilic properties, the protein network, physical states of fats and proteins, protein-polysaccharide interactions, and the gelation behavior in xanthan gum which may be responsible for the elevated textural parameters.
Xanthan gum has been in use in the food industry due to its techno-functional properties. In this study, an attempt was exerted to identify the effect of xanthan gum on the growth of probiotic bacteria. The results demonstrated that B. lactis and L. casei were capable of utilizing xanthan gum as a carbon source of fermentation comparably to the known prebiotic inulin. As a result of xanthan fermentation via bacterial enzymes, postbiotics (lactic, acetic, propionic and butyric acids) with beneficial health effects were formed. The findings of skim milk model system indicated that L. casei could remain viable in presence of xanthan gum during 28-day-storage period and there was a potential for incorporating this gum in other probiotic dairy products. The utilization of xanthan gum in skim milk matrix affected the important technofunctional parameters like acidity, syneresis, color and texture. However, these results only represent the in vitro fermentation as well as skim milk matrix. Further investigations should be carried out as in vivo model using animal and human trials and as gut model using faecal microbiota. In addition, more research is needed to prove the probiotic growth promoter effect of xanthan gum in functional foods.
This study is a part of Mervenur KANDIL’s M.Sc. thesis submitted to the Bursa Uludag University, Institute of Natural Sciences.
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Table 1. TPY and MRS Broth used as the basal medium
|
Carbohydrate-free TPY broth |
Carbohydrate-free MRS broth |
||
|
Components |
g L-1 |
Components |
g L-1 |
|
Yeast extract |
2.50 |
Yeast extract |
5.00 |
|
Peptone from meat |
5.00 |
Tryptone |
10.0 |
|
Tween 80 |
1.00 |
Peptone from meat |
10.0 |
|
K2HPO4.3H2O |
2.00 |
Tween 80 |
1.00 |
|
MgCl2 |
0.50 |
K2HPO4.3H2O |
2.00 |
|
ZnSO4.7H2O |
0.20 |
Na- acetate |
5.00 |
|
CaCl2 |
0.15 |
Ammonium citrate |
2.00 |
|
FeCl3.6H2O |
0.003 |
MgSO4.7H2O |
0.20 |
|
L-cysteine HCl |
0.50 |
MnSO4.4H2O |
0.05 |
Table 2. The mean values of pH and OD values obtained by B. lactis and L. casei species grown on different carbon sources during 48-h fermentation
|
Carbon sources |
N |
pH |
OD |
|
Control |
30 |
6,53a |
0,311d |
|
Glucose (0.50 %) |
30 |
5,05c |
1,075a |
|
Inulin (0.50 %) |
30 |
6,24b |
0,675c |
|
Xanthan gum (0.25%) |
30 |
6,38ab |
0,702c |
|
Xanthan gum (0.50%) |
30 |
6,39a |
0,915b |
|
Strains |
|||
|
B. lactis |
15 |
6,14a |
0,591b |
|
L. casei |
15 |
6,05b |
1,025a |
|
Fermantation time (hour) |
|||
|
0 |
30 |
6,53a |
0,258d |
|
12 |
30 |
6,15b |
0,774c |
|
24 |
30 |
6,11b |
0,855b |
|
36 |
30 |
5,95bc |
0,860ab |
|
48 |
30 |
5,86c |
0,931a |
|
ANOVA |
|||
|
Carbon sources |
** |
** |
|
|
Strains |
** |
** |
|
|
Fermentation time |
** |
** |
|
|
Carbon sources x Strains |
** |
** |
|
|
Carbon sources x Fermentation time |
** |
** |
|
|
Probiotic strains x Fermentation time |
** |
** |
|
|
Carbon sources x Strains x Fermentation time |
** |
** |
|
|
Significance level, significant at P<0.01(**), different lower case on the same column indicate significant differences for different carbon sources , strains and fermentation time |
|||
Table 3. Lactic acid and total SCFAs production by B. lactis and L. casei in media with different carbon sources after 48-h-fermentation
|
Carbon sources |
N |
Lactic acid |
Total SCFA |
|
Control (without carbohydrate) |
4 |
11,180a |
0,995c |
|
Glucose (0,50%) |
4 |
2,055e |
2,582a |
|
Inulin (0,50%) |
4 |
3,568b |
1,658b |
|
Xanthan gum (0,25%) |
4 |
2,075d |
0,591d |
|
Xanthan gum (0,50%) |
4 |
2,110c |
0,582d |
|
Strains |
|||
|
B. lactis |
10 |
2,769b |
0,712b |
|
L. casei |
10 |
5,626a |
1,668a |
|
ANOVA |
|||
|
Carbon sources |
** |
** |
|
|
Strains |
** |
** |
|
|
Carbon sources x strains |
** |
** |
|
|
Significance level, significant at P<0.01(**), different lower case on the same column indicate significant differences for different carbon sources, strains and fermentation time |
|||
Table 4 Viable cell counts of B. lactis and L. casei in skim milk model system during storage
|
Samples |
Storage days |
VPI |
||||
|
1. day |
7. day |
14. day |
21. day |
28. day |
||
|
BL |
9,44±0,000bB |
9,65±0,658aA |
9,00±0,001aD |
9,23±0,002aC |
8,29±0,014cE |
0,88b |
|
BLG |
9,30±0,005cA |
9,70±0,785aA |
9,40±0,636aA |
9,60±0,001aA |
8,05±0,001dA |
0,87b |
|
LC |
9,90±0,007aA |
9,80±0,289aA |
9,81±0,389aA |
9,65±0,353aA |
9,98±0,028aA |
1,01a |
|
LCG |
9,47±0,014bA |
10,10±0,572aA |
9,80±0,282aA |
9,70±0,001aA |
9,66±0,028bA |
1,02a |
|
a-dDifferent lowercase superscripts in the same column indicate the significant differences among samples for the same storage days (P<0.01) A-DDifferent uppercase superscripts in the same row indicate the significant differences among storage days for the same sample (P<0.01) BL: Skim milk with B. lactis; BLG: Skim milk with B. lactis and 0.25% xanthan gum LC: Skim milk with L. casei; LCG: Skim milk with L. casei and 0.25% xanthan gum |
||||||
Table 5 The titratable acidity, syneresis and color values of skim milk model systems during storage
|
Properties |
Samples |
Storage days |
||||
|
1. day |
7. day |
14. day |
21. day |
28. day |
||
|
Titratable acidity (%) |
BL |
0,76±0,009bA |
0,73±0,009bA |
0,76±0,000cA |
0,76±0,016bA |
0,67±0,014cB |
|
BLG |
0,81±0,005bA |
0,74±0,022bA |
0,70±0,000dA |
0,78±0,009bA |
0,82±0,055bA |
|
|
LC |
0,91±0,016aE |
1,07±0,041aD |
1,16±0,009aC |
1,20±0,005aB |
1,28±0,022aA |
|
|
LCG |
0,95±0,036aD |
1,07±0,013aC |
1,07±0,014bC |
1,16±0,063aB |
1,29±0,059aA |
|
|
Syneresis |
BL |
10,00±0,000bA |
10,00±0,354aA |
10,00±0,000bA |
10,75±0,453aA |
9,50±0,000bA |
|
BLG |
2,25±0,353cA |
2,00±0,000bA |
0,00±0,000cB |
0,00±0,212bB |
0,00±0,000cB |
|
|
LC |
12,00±0,707aA |
9,25±0,000aC |
11,50±0,000aA |
11,25±0,253aA |
10,00±0,000aB |
|
|
LCG |
0,75±0,353dA |
0,00±0,000cB |
0,00±0,000cB |
0,35±0,000bA |
0,00±0,000cB |
|
|
L* |
BL |
95,51±1,104aA |
72,69±1,256bB |
72,69±0,507aB |
72,25±0,918bB |
71,06±0,987bB |
|
BLG |
55,11±2,368dB |
62,40±1,060dA |
61,06±0,307cA |
61,64±1,205dA |
62,27±0,478cA |
|
|
LC |
76,78±1,087bA |
75,80±0,384aB |
74,76±1,839aB |
74,59±0,230aB |
73,79±1,066aB |
|
|
LCG |
61,65±0,121cC |
66,87±0,281cA |
66,43±0,136bA |
65,80±0,240cAB |
64,71±1,164cB |
|
|
a* |
BL |
-1,02±0,000cB |
-2,01±0,510bA |
-1,78 ±0,072cA |
-1,70±0,141cA |
-2,10±0,529bA |
|
BLG |
-2,47±0,442bB |
-3,90±0,042aA |
-3,98± 0,080aA |
-3,88±0,031aA |
-3,97±0,000aA |
|
|
LC |
-2,16±0,070bA |
-1,73±0,046bB |
-1,88±0,095cAB |
-1,96±0,010bAB |
-1,89±0,145bAB |
|
|
LCG |
-3,08±0,020aD |
-3,44±0,044aC |
-3,60±0,029bC |
-3,89±0,079aB |
-4,09±0,114aA |
|
|
b* |
BL |
3,66±0,350bC |
8,39±0,345aAB |
8,50±0,214aAB |
8,00±0,523aB |
9,19±0,459aA |
|
BLG |
1,15±0,142dB |
4,50±0,286cA |
4,10±0,199cA |
4,39±0,323cA |
4,56±0,185dA |
|
|
LC |
5,81±0,382aB |
6,88±0,295bAB |
7,28±0,754bA |
7,19±0,417aA |
7,15±0,325bA |
|
|
LCG |
1,94±0,012cD |
4,42±0,031cC |
4,88±0,085cC |
5,53±0,159bB |
6,11±0,398cA |
|
|
a-dDifferent lowercase superscripts in the same column indicate the significant differences among samples for the same storage days (P<0.01) A-DDifferent uppercase superscripts in the same row indicate the significant differences among storage days for the same sample (P<0.01) BL: Skim milk with B. lactis; BLG: Skim milk with B. lactis and 0.25% xanthan gum;LC: Skim milk with L. casei; LCG: Skim milk with L. casei and 0.25% xanthan gum |
||||||
Table 6. Pearson’s correlation coefficients among all the techno-functional attributes
|
|
Probiotic cell count |
Titratable acidity |
Syneresis |
L* |
a* |
b* |
Firmness |
Consistency |
Cohesiveness |
Index of viscosity |
|
Probiotic cell count |
1 |
|
|
|
|
|
|
|
|
|
|
TA |
0.998** |
1 |
|
|
|
|
|
|
|
|
|
Syneresis |
0.011 |
-0.014 |
1 |
|
|
|
|
|
|
|
|
L* |
0.069 |
0.066 |
0.950* |
1 |
|
|
|
|
|
|
|
a* |
-0.085 |
-0.100 |
0.986* |
0.973* |
1 |
|
|
|
|
|
|
b* |
0.011 |
0.010 |
0.943 |
0.998** |
0.975* |
1 |
|
|
|
|
|
Firmness |
0.494 |
0.548 |
-0.604 |
-0.353 |
-0.558 |
-0.363 |
1 |
|
|
|
|
Consistency |
0.399 |
0.459 |
-0.031 |
0.276 |
0.057 |
0.276 |
0.787 |
1 |
|
|
|
Cohesiveness |
0.540 |
0.481 |
0.145 |
-0.086 |
-0.023 |
-0.139 |
-0.333 |
-0.550 |
1 |
|
|
Index of viscosity |
-0.218 |
-0.265 |
-0.414 |
-0.678 |
-0.510 |
-0.685 |
-0.405 |
-0.883 |
0.560 |
1 |
|
Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level |
||||||||||
No competing interests reported.