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The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii ssp. bulgaricus

Open AccessPublished:August 30, 2022DOI:https://doi.org/10.3168/jds.2022-21971

      ABSTRACT

      In this study, we developed and optimized a growth medium using various nitrogen sources for the cultivation of Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and essential dairy starter culture. The composition of de Man, Rogosa, and Sharpe (MRS) culture medium was modified, and the nitrogen content was replaced by alternative nitrogen sources X-Seed Nucleo Max, X-Seed KAT, and X-Seed Carbo Max (Ohly GmbH) in various blends of 5 and 10 g/L. Results showed that bacterial growth was significantly higher when the nitrogen source blend of 10 g/L of KAT and 10 g/L of Carbo Max [KCMax (10/10)] was used. The optical densities of the Lb. bulgaricus strains were significantly higher in the KCMax (10/10) medium than in the MRS medium. There was no significance in bacterial counts for both the MRS and the KCMax (10/10) medium, and all bacterial counts were estimated at 8 log cfu/mL. The buffering capacity of the KCMax (10/10) medium was also tested and supplemented with l-histidine and was significantly higher than that of the MRS control medium. KCMax (10/10) also supported the freeze-stability and viability of the Lb. bulgaricus cells during freezing and freeze-drying operations. Our results suggest that the alternative nitrogen sources X-Seed Nucleo Max, X-Seed KAT and X-Seed Carbo Max can substantially support the growth of lactic acid bacteria as demonstrated with Lb. bulgaricus. These alternative nitrogen sources could thus be recommended for lactic acid bacteria fermentation and for the cultivation of dairy starter cultures.

      Key words

      INTRODUCTION

      Lactobacillus delbrueckii ssp. bulgaricus is an important member of the uniquely diverse group of lactic acid bacteria (LAB;
      • Gyawali R.
      • Oyeniran A.
      • Zimmerman T.
      • Aljaloud S.O.
      • Krastanov A.
      • Ibrahim S.A.
      A comparative study of extraction techniques for maximum recovery of β-galactosidase from the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus..
      ) that has many industrial applications. The LAB are generally used as starter cultures for the production of fermented dairy products such as yogurt and are also considered as probiotics due to their significant human health benefits (
      • Aponte M.
      • Murru N.
      • Shoukat M.
      Therapeutic, prophylactic, and functional use of probiotics: A current perspective.
      ). Lactobacillus delbrueckii ssp. bulgaricus is one of the 2 bacteria required for commercial yogurt production and is used synergistically with Streptococcus thermophilus on an industrial scale for the production of yogurt. This yogurt bacterium plays a vital role in the development of the organoleptic (
      • Petry S.
      • Furlan S.
      • Crepeau M.J.
      • Cerning J.
      • Desmazeaud M.
      Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium.
      ) and probiotic properties of yogurt (
      • Hayek S.A.
      • Gyawali R.
      • Aljaloud S.O.
      • Krastanov A.
      • Ibrahim S.A.
      Cultivation media for lactic acid bacteria used in dairy products.
      ;
      • Ayivi R.D.
      • Gyawali R.
      • Krastanov A.
      • Aljaloud S.O.
      • Worku M.
      • Tahergorabi R.
      • Claro da Silva R.
      • Ibrahim S.A.
      Lactic Acid Bacteria: Food Safety and Human Health Applications.
      ). Moreover, it has been reported to be a safe probiotic with several health benefits when administered in adequate amounts at an effective dose (
      • Adolfsson O.
      • Meydani S.N.
      • Russell R.M.
      Yogurt and gut function.
      ). The flavor, texture, and organoleptic properties of yogurt are generally a result of the symbiotic interaction of Lb. bulgaricus and Strep. thermophilus. Consequently, an imbalance in these 2 bacteria will affect the sensorial characteristics and qualities of yogurt as a fermented dairy product (
      • Sieuwerts S.
      Microbial interactions in the yoghurt consortium: Current status and product implications.
      ).
      Lactobacillus delbrueckii ssp. bulgaricus as a starter culture is very vital in many fermentation and bioprocessing operations. The fermentation performance and functionality of most strains of Lb. bulgaricus is enhanced by nutritional requirements and the overall enrichment of the fermentation medium (
      • Ayivi R.D.
      • Gyawali R.
      • Krastanov A.
      • Aljaloud S.O.
      • Worku M.
      • Tahergorabi R.
      • Claro da Silva R.
      • Ibrahim S.A.
      Lactic Acid Bacteria: Food Safety and Human Health Applications.
      ). The food and dairy industries are continually in search of various probiotic bacteria for applications with cost-effective fermentation processes including growth medium requirements (
      • Akabanda F.
      • Owusu-Kwarteng J.
      • Tano-Debrah K.
      • Glover R.L.
      • Nielsen D.S.
      • Jespersen L.
      Taxonomic and molecular characterization of lactic acid bacteria and yeasts in nunu, a Ghanaian fermented milk product.
      ;
      • Hayek S.A.
      • Ibrahim S.A.
      Current limitations and challenges with lactic acid bacteria: A review.
      ;
      • Mani-López E.
      • Palou E.
      • Lopez-Malo A.
      Probiotic viability and storage stability of yogurts and fermented milks prepared with several mixtures of lactic acid bacteria.
      ;
      • Atilola O.A.
      • Gyawali R.
      • Aljaloud S.O.
      • Ibrahim S.A.
      Use of phytone peptone to optimize growth and cell density of Lactobacillus reuteri..
      ). Generally, the nutritional quality of the fermentation medium is enhanced by the supplementation of casein hydrolysates and yeast extract, which are key nitrogen sources that act as growth-promoters for boosting high bacterial cell growth (
      • Norton S.
      • Lacroix C.
      • Vuillemard J.C.
      Kinetic study of continuous whey permeate fermentation by immobilized Lactobacillus helveticus for lactic acid production.
      ;
      • Hayek S.A.
      • Ibrahim S.A.
      Current limitations and challenges with lactic acid bacteria: A review.
      ). Significant biomass levels and high volumes of lactic acid production by LAB species have also been attributed to the presence of AA, vitamins and, yeast extract in the fermentation medium (
      • Alazzeh A.Y.
      • Ibrahim S.A.
      • Song D.
      • Shahbazi A.
      • AbuGhazaleh A.A.
      Carbohydrate and protein sources influence the induction of α-and β-galactosidases in Lactobacillus reuteri..
      ;
      • Manzoor A.
      • Qazi J.I.
      • Haq I.
      • Mukhtar H.
      • Rasool A.
      Significantly enhanced biomass production of a novel bio-therapeutic strain Lactobacillus plantarum (AS-14) by developing low-cost media cultivation strategy.
      ;
      • Ayad A.A.
      • Gad El-Rab D.A.
      • Ibrahim S.A.
      • Williams L.L.
      Nitrogen sources effect on Lactobacillus reuteri growth and performance cultivated in date palm (Phoenix dactylifera L.) by-products.
      ). The freeze-drying of LAB cells is also highly dependent on the growth medium, fermentation pH, cryoprotectant and other intrinsic factors (
      • Carvalho A.S.
      • Silva J.
      • Ho P.
      • Teixeira P.
      • Malcata F.X.
      • Gibbs P.
      Relevant factors for the preparation of freeze-dried lactic acid bacteria.
      ). This characteristic has been confirmed by a study by
      • Chen H.
      • Huang J.
      • Shi X.
      • Li Y.
      • Liu Y.
      Effects of six substances on the growth and freeze-drying of Lactobacillus delbrueckii subsp. bulgaricus..
      in which a supplemented MRS medium significantly affected the growth and freeze-drying viability of Lb. bulgaricus strains. An enriched growth medium has also been credited with improving the freeze-drying survival rate of LAB due to the medium's composition (carbon, nitrogen, and lipids such as Tween 80), which efficiently helps to reduce bacterial stress during the freeze-drying process (
      • Li C.
      • Liu L.B.
      • Liu N.
      Effects of carbon sources and lipids on freeze-drying survival of Lactobacillus bulgaricus in growth media.
      ).
      • Chervaux C.
      • Ehrlich S.D.
      • Maguin E.
      Physiological study of Lactobacillus delbrueckii ssp. bulgaricus strains in a novel chemically defined medium.
      confirmed that Lb. bulgaricus cultivated in a growth medium highly supplemented with different carbon sources enhanced the stress resistance of the bacteria during various treatments. Interestingly, Tween 80 supplemented in a growth medium transformed the fatty acid composition of the LAB cells after fermentation, significantly enhancing the resistance of the cells to freezing and freeze-drying (
      • Carvalho A.S.
      • Silva J.
      • Ho P.
      • Teixeira P.
      • Malcata F.X.
      • Gibbs P.
      Relevant factors for the preparation of freeze-dried lactic acid bacteria.
      ).
      However, the limiting factor for supplementing fermentation media with high levels of nitrogen sources stems from the astronomical cost (
      • Manzoor A.
      • Qazi J.I.
      • Haq I.
      • Mukhtar H.
      • Rasool A.
      Significantly enhanced biomass production of a novel bio-therapeutic strain Lactobacillus plantarum (AS-14) by developing low-cost media cultivation strategy.
      ;
      • Hayek S.A.
      • Gyawali R.
      • Aljaloud S.O.
      • Krastanov A.
      • Ibrahim S.A.
      Cultivation media for lactic acid bacteria used in dairy products.
      ). Moreover, the standard LAB fermentation medium, de Man, Rogosa, and Sharpe (MRS), is expensive due to its nitrogen sources (meat, peptone, and yeast extract) and does not support the growth of all LAB and probiotic cultures. The available literature also suggests that significant research is aimed at finding alternative cost-effective ingredients for LAB growth media, as well as obtaining higher cell densities and biomass levels. Therefore, the objectives of the present study were to investigate the growth and viability of Lb. bulgaricus cultivated with alternative nitrogen sources and to assess the impact on the viability of Lb. bulgaricus after freeze-drying.

      MATERIALS AND METHODS

      Source of Lb. bulgaricus and Preparation of Preculture Fermentation Medium

      A total of 10 Lb. bulgaricus strains were used for the preliminary study (not all data are shown) as elucidated in Table 1 with the original source. Strains (S9 and LB6) were selected in this study based on different growth rates (
      • Georgiev M.G.
      • Goranov B.
      • Ilyazova A.
      • Ibrahim S.
      • Krastanov A.
      Solid-phase micro extraction coupled with GC–MS for determination of the metabolite profile of newly isolated strains of Lactobacillus delbrueckii ssp. bulgaricus..
      ). Strains were activated in MRS as the prefermentative medium for the Lb. bulgaricus strains as detailed in the Supplemental File S1 (https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ). Enumeration of the evaluated strains was done using MRS agar and modified reinforced clostridial medium-pyruvate (mRCM-PYR) with details of their preparation described in the Supplemental File S1. No animals were used in this study, and ethical approval for the use of animals was thus deemed unnecessary.
      Table 1Probiotic strains used in the preliminary study
      Product codeSampleOriginal sourceBacterial composition as labeled
      S9Pure industrial strainBulgariaLactobacillus bulgaricus
      LB6Pure industrial strainBulgariaLb. bulgaricus,
      THTPure industrial strainBelgiumLb. bulgaricus
      ATCC 11842Pure industrial strainATCCLb. bulgaricus
      DAWYogurtUSALb. bulgaricus, other live culture
      S8Pure industrial strainBulgariaLb. bulgaricus
      E22YogurtUSALb. bulgaricus, other live culture
      S5Pure industrial strainBulgariaLb. bulgaricus
      S19Pure industrial strainBulgariaLb. bulgaricus
      GenesisProbiotic supplementBulgariaLb. bulgaricus

      Validation of Nitrogen Sources, Optimization of Growth Medium, and Culture Propagation in Optimized Growth Medium

      Five yeast samples [X-Seed Nucleo Max (N-Max), X-Seed KAT (KAT), X-Seed Peptone (PEP), X-Seed Carbo Peptone (C-Pep), and X-Seed Carbo Max (C-Max)] provided by Ohly GmbH were evaluated in different blends and proportions during the preliminary study. Both C-Pep and C-Max are developmental products, and as such their description is proprietary and not available. The preliminary work thus confirmed N-Max, KAT, and C-Max yeast samples as superior alternative complex nitrogen sources for use in the study. Supplemental Table S1 (https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ) highlights the yeast description, total percentage of protein, and total percentage of nitrogen in the N-Max, KAT, and C-Max, respectively. The optimized growth medium was prepared similarly to the composition of MRS (Supplemental Table S2; https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ); however, its major nitrogen sources were replaced with N-Max, and other optimized proportions of blends of KAT, and C-Max as alternative nitrogen sources (Supplemental Table S3; https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ). The 2 validated strains (S9 and LB6) were then propagated in the optimized growth medium per the formulations in Supplemental Table S4 (https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ). Details of the culture propagation procedure are highlighted in the supplemental file.

      Determination of pH Values and Buffering Capacity of Growth Medium with l-Arginine and l-Histidine Supplementation

      The pH values of each medium were measured in duplicates at the start and end of fermentation. The buffering capacity of all media was also evaluated with 4 g/L of l-arginine and l-histidine, respectively. Supplemental File S1 highlights the pH and the buffer capacity determination.

      Bacterial Enumeration and Effect of Growth Media on the Freeze-Stability and Viability of Lb. bulgaricus.

      The bacterial growth of the 2 strains (LB6 and S9) was monitored via spectrophotometric measurement of their optical densities (OD610nm) at different time intervals (0, 6, and 12 h) in the optimized media. All strains were enumerated at the end of the fermentation period (12 h), by the surface-plated method as detailed in the supplemental file. The S9 Lb. bulgaricus strain demonstrated higher cell growth than the LB6 strain and as such was chosen and evaluated for viability after freezing and freeze-drying with and without recovery treatment as detailed in the Supplemental File S1. All plates were then anaerobically incubated for 48 h at 42°C and plates with 25 to 250 colonies were counted. Bacterial populations were expressed in log colony-forming units per mililiter (Table 2).
      Table 2Bacterial count (mean ± SD; n = 3) of Lactobacillus bulgaricus strains expressed as log cfu/mL after 12 h in different growth media
      Growth medium
      MRS = de Man, Rogosa, and Sharpe medium (Neogen Corporation). Ohly GmbH is manufacturer of all of the nitrogen source blends: N-Max = X-Seed Nucleo Max; C-Max = X-Seed Carbo Max; KAT = X-Seed KAT; KCMax = a blend of both KAT and C-Max. The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max, and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max.
      S9 strainLB6 strain
      MRS7.63 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      7.06 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      N-Max (basal medium)5.0 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      5.0 ± 0.14
      Means with different superscripts within a row differ significantly (P < 0.05).
      C-Max 5%5.0 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      5.15 ± 0.21
      Means with different superscripts within a row differ significantly (P < 0.05).
      C-Max 10%5.24 ± 0.34
      Means with different superscripts within a row differ significantly (P < 0.05).
      5.0 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      KAT 5%6.04 ± 0.06
      Means with different superscripts within a row differ significantly (P < 0.05).
      7.08 ± 0.0
      Means with different superscripts within a row differ significantly (P < 0.05).
      KAT 10%6.74 ± 0.06
      Means with different superscripts within a row differ significantly (P < 0.05).
      7.16 ± 0.02
      Means with different superscripts within a row differ significantly (P < 0.05).
      KCMax (5/5)6.49 ± 0.04
      Means with different superscripts within a row differ significantly (P < 0.05).
      6.53 ± 0.04
      Means with different superscripts within a row differ significantly (P < 0.05).
      KCMax (5/10)7.32 ± 0.01
      Means with different superscripts within a row differ significantly (P < 0.05).
      6.31 ± 0.01
      Means with different superscripts within a row differ significantly (P < 0.05).
      KCMax (10/5)6.97 ± 0.02
      Means with different superscripts within a row differ significantly (P < 0.05).
      6.64 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      KCMax (10/10)7.22 ± 0
      Means with different superscripts within a row differ significantly (P < 0.05).
      6.49 ± 0.02
      Means with different superscripts within a row differ significantly (P < 0.05).
      a–d Means with different superscripts within a row differ significantly (P < 0.05).
      1 MRS = de Man, Rogosa, and Sharpe medium (Neogen Corporation). Ohly GmbH is manufacturer of all of the nitrogen source blends: N-Max = X-Seed Nucleo Max; C-Max = X-Seed Carbo Max; KAT = X-Seed KAT; KCMax = a blend of both KAT and C-Max. The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max, and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max.

      Statistical Analyses

      SAS version 9.4 (SAS Institute Inc.) was used to analyze the data obtained in the study, and ANOVA was used to determine significant differences between obtained values for the final optimized growth media blends but not for the preliminary study. Significant differences (P < 0.05) between treatment means were compared using Tukey's test. Bacterial population counts were expressed in log10 before analyses were made.

      RESULTS

      Preliminary Study (Validation of the Nitrogen Source on the Growth of Lb. bulgaricus Strains)

      Evaluation of the nitrogen source on the growth of the different strains of Lb. bulgaricus was done in the preliminary study. Although it was known that bacterial growth in the fermentation medium was mostly strain-dependent, it was generally observed that some Lb. bulgaricus strains possessed higher growth rates (OD610nm) than others, and therefore, their growth was significant (P < 0.05). Consequently, S9 and LB6 bulgaricus strains were selected for the study. In validating the different nitrogen sources, N-Max, KAT, PEP, C-Pep, and C-Max were tested in different blends and proportions (Supplemental Table S4) thereby confirming which complex nitrogen source could best support the growth of the Lb. bulgaricus strains. Our findings also showed that blends containing PEP and C-Pep inhibited the growth of the Lb. bulgaricus strains and were therefore not selected for the main study. As a result, N-Max, KAT, and C-Max were considered superior complex nitrogen sources and were therefore blended in different proportions for use as the final growth medium. The selected complex nitrogen sources were not unexpected. Their total nitrogen percentages were 71, 75, and 30% for N-Max, KAT, and C-Max, respectively, compared with the total nitrogen percentages of 67 and 45% of PEP, and C-Pep, respectively.
      Various blends of the different nitrogen sources were used in formulating the growth media, and these formulations were inspired based on the study design of
      • Somani S.A.
      • Boran B.
      • Bekers K.
      Impact of nucleotides on L. bulgaricus growth, viability and performance. Ohly White Paper.
      . Several trials were done with various strains of Lb. bulgaricus until a final formulation with comparable results to MRS was obtained. The first formulation had a blend of 1% (wt/vol) of the different nitrogen sources, whereby 5 strains (E22, S9, LB6, RDA, and ATCC 11842) were used. MRS was also used as a control medium in all of the preliminary formulations. The growth of the bacteria was monitored for 8 h at an incubation temperature of 42°C. Although the optical density (OD; OD610nm) results of the strains in the first formulated growth medium were not promising, MRS (the standard control) had good growth confirming the viability of the strains as shown in Supplemental Figure S1 (https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ).
      It was also observed that the Lb. bulgaricus strains S9 and LB6 were fast growing, especially in the MRS medium, hence their selection for the fermentation study. Formulations 2, and 4 (data not shown) did not yield promising growth results; however, formulation 3 (Supplemental Figure S2; https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ) included a basal medium with 2 g/L of N-Max in conjunction with various blends of KAT and C-Max yielded exceptional results.
      Based on the positive outcome of formulation 3, formulation 5 was developed and consequently chosen as the final optimized recipe for the study. The fifth and final growth medium formulation was based on 5% (5 g/L) and 10% (10 g/L) of the validated nitrogen sources (KAT and C-Max, respectively), and a blend of both KAT and C-Max (KCMax) in a supplemented basal medium containing 2 g/L of N-Max. Thus, the various formulations employed were C-Max 5%, C-Max 10%, KAT 5%, KAT 10%, KCMax (5/5), KCMax (5/10), KCMax (10/5), and KCMax (10/10), all of which were supplemented with a basal medium (2 g/L of N-Max). The MRS was also used as the control medium in all cases for the fermentation study. The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max, and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max.

      Growth and Cell Density in the Final Optimized Growth Medium

      The Lb. bulgaricus strains (S9 and LB6) used in the study demonstrated varied growth patterns in the final medium during the 12-h incubation period at 42°C. The growth medium MRS also confirmed that the strains were viable as the strains adequately utilized the nutrients inherent in the MRS medium for their cellular activity. There was a significant difference (P < 0.05) in the OD results for the blends of KAT and C-Max, with KCMax (10/10) performing better than the MRS medium after 12 h of fermentation. Figure 1 shows the growth pattern (OD610nm) of the S9 and LB6 strains in the complex nitrogen-supplemented media during 12 h of fermentation at 42°C.
      Figure thumbnail gr1
      Figure 1Changes in the growth of S9 and LB6 Lactobacillus bulgaricus strains at optical density at 610 nm (OD610nm) in the different final growth media at different proportions and blends after 12 h of fermentation at 42°C. MRS = de Man, Rogosa, and Sharpe. Basal medium: N-Max = X-Seed Nucleo Max. The basal medium supplemented with different nitrogen sources: C-Max = X-Seed Carbo Max; KAT = X-Seed KAT; KCMax = blend of both KAT and C-Max. Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation. Error bars indicate SD for an experiment performed in duplicate; different lowercase letters indicate statistical differences between the various growth media.
      There was also a significant difference (P < 0.05) in the OD results of the blends of KAT and C-Max media in reference to the other growth media. The observed growth of S9 and LB6 in the different media suggests the availability of complex nitrogen in the formulated medium was vital for microbial growth. The basal medium alone for both S9 and LB6 strains was not adequately enriched to support the metabolic activities of the Lb. bulgaricus strains; hence, the observed poor growth result. The bacterial growth in MRS and KAT and KCMax media thus suggested that bacterial growth was affected by the nitrogen content in the media.

      Enumeration of Lb. bulgaricus Strains

      There was a significant difference (P < 0.05) in the bacterial counts between the basal medium (N-Max), C-Max 5%, C-Max 10%, KAT 5%, KAT 10%, and the different blends of KCMax media. However, no significant difference in the bacterial counts of the MRS medium and the KCMax (5/10), KCMax (10/5), and the KCMax (10/10) were recorded. Although bacterial growth performance is mostly strain-dependent, the observed bacterial counts especially from the blends of nitrogen sources KAT and C-Max presupposes that these media can be compared with the performance of MRS as a growth medium and could be selected as alternative media for supporting the growth of Lb. bulgaricus. The bacterial counts of the strains (S9 and LB6) are presented in Table 1.

      pH Value Changes in the Different Growth Media

      The initial pH value of all of the media was adjusted to 6.5 before the start of the fermentation process using the S9 and LB6 strains. However, at the end of the 12-h fermentation period for the S9 strain, there was a rapid decline in pH from the initial value of 6.5 to a final value between 5.33 and 5.62 for MRS, KAT, and blends of KAT and C-Max. This rapid decline in pH was similarly observed for the LB6 strain whereby the final pH values after fermentation were in the range of 5.06 to 5.72 for the MRS control medium, KAT, and the blends of KAT and C-Max media. Therefore, a significant difference (P < 0.05) was observed for the formulated media with different complex nitrogen sources in comparison to the MRS (control) medium. Interestingly, the observed pH results for the strains (S9 and LBS) for the N-Max and C-Max media following the fermentation period were in contrast to that observed in the MRS, KAT, and the blends of KAT and C-Max. Thus, the final pH values of the S9 and LB6 strains for N-Max and C-Max were in the range of 6.26 to 6.36 and 6.14 to 6.38, respectively. Changes in pH of the various media are depicted in Figure 2. The observed differences in pH values could be attributed to the presence of the alternative protein sources in the various media, which resulted in a buffering potential and thus restricted changes in pH. To determine this effect, an additional study related to the buffering capacity of the different protein sources was performed.
      Figure thumbnail gr2
      Figure 2Changes in pH of the Lactobacillus bulgaricus strains (S9 and LB6) in the control medium de Man, Rogosa, and Sharpe (MRS), basal medium X-Seed Nucleo Max (N-Max), and the basal medium supplemented with different nitrogen sources X-Seed KAT (KAT) X-Seed Carbo Max (C-Max) at different proportions and blends after 12 h of fermentation. Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation. Error bars indicate SD for an experiment performed in duplicate; different lowercase letters indicate statistical differences between the various growth media.

      Buffering Capacity of Growth Media with l-Arginine and l-Histidine Supplementation

      Buffering capacity of a growth media is an important fermentation parameter, as it provides a stable pH range for the metabolic activity of LAB. This stable pH, promoted high cell density of bacterial growth, as well as limiting the production of organic acid (lactic acid), which could cause cell injury thereby decreasing the growth of the strains. The buffering capacity of the growth medium was evaluated with 2 essential AA (l-arginine and l-histidine). The change in buffering capacity was highly observed with the supplementation of l-histidine than with l-arginine. Three growth media blends, KCMax (5/10), KCMax (10/5), and KCMax (10/10), had the highest significant buffering capacity values of 36, 36.5, and 39, respectively (Figure 3, Figure 4), with the l-histidine supplementation (P < 0.05) as compared with the buffering capacity of the standard l-histidine supplemented MRS medium (35.22; Table 3). Similarly, these 3 KCMax media blends also reported high buffering capacity values (15.24, 15.24, and 15.50, respectively) without any l-arginine or l-histidine supplementation but were, however; lower than the value reported in the standard MRS medium (21.57; Table 3). When the growth media was supplemented with l-arginine, no significant difference (P > 0.05) was observed in all cases for the blends, as the buffering capacity values were almost the same as that of the normal media without any AA supplementation. Therefore, l-arginine supplementation in the growth media did not have any significant effect on the media's buffering capacity. It was, therefore, evident that the KCMax (10/10) medium had the best buffering capacity (39) with the supplementation of l-histidine as compared with the standard MRS medium (35.22). A higher buffering capacity value, therefore, suggested the medium was well suited to promote higher cell density of LAB growth during fermentation operations. Consequently, bacterial growth and metabolic activity are sustained to a high level, therefore promoting better fermentation processes. Generally, for LAB fermentation, it is recommended to consider a growth medium that provides a high pH buffering. Table 3 summarizes the buffering capacity for the different growth media with the supplementation of l-arginine and l-histidine.
      Figure thumbnail gr3
      Figure 3The different buffering capacities of (a) KCMax (5/5) and (b) KCMax (5/5) supplemented with l-histidine (His), and (c) KCMax (5/10) and (d) KCMax (5/10) medium supplemented with His. KCMax = a blend of both X-Seed KAT (KAT) and X-Seed Carbo Max (C-Max). The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max. Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation.
      Figure thumbnail gr4
      Figure 4The varying buffering capacities of (a) KCMax (10/5) and (b) KCMax (10/5) supplemented with l-histidine (His), and (c) KCMax (10/10) and (d) KCMax (10/10) medium supplemented with His. KCMax = a blend of both X-Seed KAT (KAT) and X-Seed Carbo Max (C-Max). The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max, and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max. Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation.
      Table 3Buffering capacity of the different growth media with and without l-arginine and l-histidine supplementation
      Growth medium
      MRS = de Man, Rogosa, and Sharpe; N-Max = X-Seed Nucleo Max; C-Max = X-Seed Carbo Max; KAT = X-Seed KAT; KCMax = a blend of both KAT and C-Max. Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation. The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max, and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max.
      Buffer capacity value (no supplementation)Buffer capacity value (arginine supplementation)Buffer capacity value (histidine supplementation)
      MRS21.5721.8435.22
      N-Max (Basal)13.051330
      C-Max 5%12.9112.1731
      C-Max 10%13.1513.2534
      KAT 5%12.9713.0432
      KAT 10%14.0714.3334.5
      KCMax (5/5)15.0514.0333
      KCMax (5/10)15.2415.036
      KCMax (10/5)15.2414.536.5
      KCMax (10/10)15.5016.2539
      1 MRS = de Man, Rogosa, and Sharpe; N-Max = X-Seed Nucleo Max; C-Max = X-Seed Carbo Max; KAT = X-Seed KAT; KCMax = a blend of both KAT and C-Max. Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation. The nitrogen source blends KCMax (5/5) represented 5 g/L of KAT and 5 g/L of C-Max, and KCMax (5/10) was a combination of 5 g/L of KAT and 10 g/L of C-Max. The blend KCMax (10/5) consisted of 10 g/L of KAT and 5 g/L of C-Max, and the final blend KCMax (10/10) was a combination of 10 g/L of KAT and 10 g/L of C-Max.
      The l-histidine supplementation provided a better indication of the pH buffering capacity of the media and was thus very promising, having a stable resistance to pH change over a longer period with higher consumption of the 0.1 N HCl solution. There was a significant difference (P < 0.05) in the buffering capacity values of the l-histidine supplemented media compared with that of the l-arginine supplemented and the nonsupplemented growth media. The use of a minimal amount of this EAA proved effective in maintaining a stable pH over some time, ensuring a good buffering for higher cell growth (cfu/mL). The observed results confirmed l-histidine as a potential natural alternative that could be used for maintaining a stable fermentation pH in fermentation cultures for pH buffering of growth media. Supplemental Figures S3 to S10 (https://doi.org/10.17632/hzx7t8htdp.1;
      • Ayivi R.
      • Ibrahim S.
      • Krastanov A.
      • Somani A.
      • Siddiqui S.A.S.
      Supplemental Files. “The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii subsp. bulgaricus.” Mendeley Data, V1.
      ) highlight the different pH buffering of the growth media with and without the supplementation of the 2 AA (l-arginine and l-histidine), respectively.

      Effect of the Optimized Growth Media (KCMax 10/10) on the Freeze-Stability and Viability of Lb. delbrueckii ssp. bulgaricus

      The initial count of the MRS medium strain was 8.5 log cfu/mL and that for the KCMax (10/10) medium was 8.4 log cfu/mL. The population count for the MRS cultivated strain after overnight freezing at −80°C was 8.8 log cfu/mL. Interestingly, the population count for the KCMax (10/10) cultivated strain after overnight freezing was a bit higher (9.1 log cfu/mL) than the MRS cultivated strain. There was, however, no significant difference (P > 0.05) between the counts of the MRS and the KCMax (10/10) medium after the freezing process. The count of the freeze-dried bulgaricus cells cultivated from the MRS medium decreased compared with the initial count and was 5.8 log cfu/mL. A similar observation of reduced viability (5.2 log cfu/mL) was reported for the freeze-dried bulgaricus cells cultivated from the KCMax (10/10) medium. However, there was a much higher increase in cell counts for the freeze-dried cells after the cells were recovered in skim milk which resulted in the resuscitation and repair of the stressed and injured cells after the freeze-drying process. The population count for the MRS recovered freeze-dried cells was 6.5 log cfu/mL. Consequently, the population count for the KCMax (10/10) recovered freeze-dried cells was higher than what was observed in the MRS (standard) at 7.3 log cfu/mL. Figure 5 elucidates the viability of Lb. bulgaricus in both the standard (MRS) and the KCMax (10/10) media after the freeze-drying process.
      Figure thumbnail gr5
      Figure 5Viability and bacterial count of S9 Lactobacillus bulgaricus strain before and after freezing and freeze-drying with and without recovery after fermentation in de Man, Rogosa, and Sharpe (MRS) and KCMax (10/10) medium. KCMax (10/10) = a blend of 10 g/L X-Seed KAT (KAT) and 10 g/L X-Seed Carbo Max (C-Max). Ohly GmbH is the manufacturer of all the nitrogen sources; MRS is from Neogen Corporation. Error bars indicate SD for an experiment performed in duplicate; different lowercase letters indicate statistical differences between the various growth media.

      DISCUSSION

      Several studies have evaluated the effect of commercial nitrogen sources in various selective growth media for the enhanced growth promotion of LAB cultures (
      • Aeschlimann A.
      • von Stockar U.
      The effect of yeast extract supplementation on the production of lactic acid from whey permeate by Lactobacillus helveticus..
      ;
      • Champagne C.P.
      • Gaudreau H.
      • Conway J.
      • Chartier N.
      • Fonchy E.
      Evaluation of yeast extracts as growth media supplements for lactococci and lactobacilli by using automated spectrophotometry.
      ;
      • Gaudreau H.
      • Champagne C.P.
      • Conway J.
      • Degré R.
      Effect of ultrafiltration of yeast extracts on their ability to promote lactic acid bacteria growth.
      ;
      • Atilola O.A.
      • Gyawali R.
      • Aljaloud S.O.
      • Ibrahim S.A.
      Use of phytone peptone to optimize growth and cell density of Lactobacillus reuteri..
      ). In the present study, we modified the composition of MRS and evaluated the effect of different nitrogen sources (N-Max, KAT, and C-Max) in an optimized medium on the growth and performance of Lb. bulgaricus strains. Yeast extracts are excellent sources of vitamins, AA, and peptides and have been credited with the enhanced stimulatory growth of LAB cultures when employed in culture medium (
      • Juillard V.
      • Le Bars D.
      • Kunji E.R.
      • Konings W.N.
      • Gripon J.C.
      • Richard J.
      Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk.
      ;
      • Champagne C.P.
      • Gaudreau H.
      • Conway J.
      • Chartier N.
      • Fonchy E.
      Evaluation of yeast extracts as growth media supplements for lactococci and lactobacilli by using automated spectrophotometry.
      ). Furthermore, the major criteria for enhanced viability and high yields of dairy starter cultures industrially have been characterized by the nitrogen component of the culture medium utilized for their production (
      • Proust L.
      • Sourabié A.
      • Pedersen M.
      • Besançon I.
      • Haudebourg E.
      • Monnet V.
      • Juillard V.
      Insights into the complexity of yeast extract peptides and their utilization by Streptococcus thermophilus..
      ).
      The strain-dependent characteristics of Lactobacillus species for nutritional requirements warrant several optimization techniques to promote bacterial growth and achieve high cell density (
      • Ren H.
      • Zentek J.
      • Vahjen W.
      Optimization of production parameters for probiotic Lactobacillus strains as feed additive.
      ). The alternative yeast extracts (N-Max, KAT, and C-Max) in the modified growth media were highly efficient and showed promising results in comparison to the standard and conventional MRS medium. The effect of yeast extract on the growth of LAB cultures was also corroborated by
      • Zhang J.
      • Bu Y.
      • Zhang C.
      • Yi H.
      • Liu D.
      • Jiao J.
      Development of a low-cost and high-efficiency culture medium for Bacteriocin Lac-B23 production by Lactobacillus plantarum J23.
      in their study when a modified MRS medium was enhanced with yeast extract to promote bacteriocin production. Another study by
      • Abbasiliasi S.
      • Ramanan R.N.
      • Ibrahim T.A.T.
      • Mustafa S.
      • Mohamad R.
      • Daud H.H.M.
      • Ariff A.B.
      Effect of medium composition and culture condition on the production of bacteriocin-like inhibitory substances (BLIS) by Lactobacillus paracasei LA07, a strain isolated from Budu.
      also confirmed the effect of yeast extract as a nitrogen source that significantly enhanced the production of bacteriocin-like inhibitory substances in a developed growth medium for Lactobacillus paracasei LA07. The blends of KAT and C-Max in the basal medium containing N-Max yielded superior results than the MRS medium and this could be attributed to the bioavailability of free nucleotides, amino acids and peptides in the optimized medium (
      • Somani S.A.
      • Boran B.
      • Bekers K.
      Impact of nucleotides on L. bulgaricus growth, viability and performance. Ohly White Paper.
      ). We also observed that the combination of these alternative yeast extracts was responsible for the enhanced growth of the LAB strains, as the optimized medium in the absence of these nitrogen sources confirmed poor or no growth of the evaluated LAB strains. Consequently, blending and selection of appropriate nutrient-rich yeast extracts for growth medium supplementation potentially enhances the cellular activity and the growth performance of LAB cultures (
      • Somani S.A.
      • Boran B.
      • Bekers K.
      Impact of nucleotides on L. bulgaricus growth, viability and performance. Ohly White Paper.
      ;
      • Xiao S.
      • Bekers M.K.
      • van der Werf M.J.
      Using DoE to optimize yeast extract composition for lactic acid bacteria. Ohly White Paper.
      ). The enhanced superior growth at OD610nm and the cell counts for both S9 and LB6 strains in the optimized medium were also confirmed by
      • Somani S.A.
      • Boran B.
      • Bekers K.
      Impact of nucleotides on L. bulgaricus growth, viability and performance. Ohly White Paper.
      in their study when the same yeast extracts had a significant effect on the growth performance of LAB cultures. The pH and buffering capacity for the growth medium were evaluated with 2 AA (l-arginine and l-histidine); however, l-histidine was confirmed as a better alternative for enhancing the growth medium's buffering capacity, which promoted enhanced LAB growth. Buffering capacity and pH are thus very critical factors in media development as they ensure a stable pH without comprising the enhanced cellular activities of LAB cultures (
      • Hayek S.A.
      • Gyawali R.
      • Aljaloud S.O.
      • Krastanov A.
      • Ibrahim S.A.
      Cultivation media for lactic acid bacteria used in dairy products.
      ). This was also confirmed by
      • Azatian S.B.
      • Kaur N.
      • Latham M.P.
      Increasing the buffering capacity of minimal media leads to higher protein yield.
      whereby increasing a growth medium's buffering capacity led to higher protein yields in Escherichia coli. The growth medium's composition plays a vital role in minimizing viability losses during freeze-drying processes and also enhances the freeze-stability of LAB cell cultures (
      • Chervaux C.
      • Ehrlich S.D.
      • Maguin E.
      Physiological study of Lactobacillus delbrueckii ssp. bulgaricus strains in a novel chemically defined medium.
      ;
      • Carvalho A.S.
      • Silva J.
      • Ho P.
      • Teixeira P.
      • Malcata F.X.
      • Gibbs P.
      Relevant factors for the preparation of freeze-dried lactic acid bacteria.
      ;
      • Fenster K.
      • Freeburg B.
      • Hollard C.
      • Wong C.
      • Rønhave Laursen R.
      • Ouwehand A.C.
      The production and delivery of probiotics: A review of a practical approach.
      ). We also observed that the optimized growth medium (KCMax 10/10) had a significant effect on the viability of LAB after freezing and freeze-drying processes with and without cell recovery in milk. This observation could be explained due to the rich nitrogen composition, free AA, vitamins, minerals, and peptides of the yeast extract that could significantly enhance cell survival during freeze-drying (
      • Somani A.
      • Boran B.
      • Bekers K.
      Influence of growth media composition on cell stability during freeze-drying. Ohly White Paper.
      ). In addition, these yeast extracts as alternative nitrogen sources have been confirmed as not only affecting the growth of LAB cultures but also enhancing the viability and affecting cell survival during freeze-drying (
      • Somani A.
      • Boran B.
      • Bekers K.
      Influence of growth media composition on cell stability during freeze-drying. Ohly White Paper.
      ). According to a study by
      • Chervaux C.
      • Ehrlich S.D.
      • Maguin E.
      Physiological study of Lactobacillus delbrueckii ssp. bulgaricus strains in a novel chemically defined medium.
      , a growth medium supplemented with all known AA, vitamins, and micronutrients was able to efficiently support the growth and viability of Lb. bulgaricus CNRZ397 during fermentation and freeze-drying processes.
      • Carvalho A.S.
      • Silva J.
      • Ho P.
      • Teixeira P.
      • Malcata F.X.
      • Gibbs P.
      Relevant factors for the preparation of freeze-dried lactic acid bacteria.
      also confirmed the effect of AA on reinforcing the intrinsic protective properties in bacterial cell walls. This uniquely shields the cell from stress and enhances its resistance during freeze-drying processes. For the freeze-dried cells, viability was also enhanced after cell recovery in milk. The successful resuscitation of freeze-dried dairy LAB cultures in milk has been widely reported and employed for enhancing the viability of freeze-dried cells (
      • Celik O.F.
      • O'Sullivan D.J.
      Factors influencing the stability of freeze-dried stress-resilient and stress-sensitive strains of bifidobacteria.
      ). Thus, the high bacterial count observed for the recovered freeze-dried strains cultivated in the KCMax (10/10) medium could be attributed to both the nitrogen-rich profile of the yeast extracts as well as the effect of the milk nutrients (
      • Somani A.
      • Boran B.
      • Bekers K.
      Influence of growth media composition on cell stability during freeze-drying. Ohly White Paper.
      ). The KCMax (10/10) medium could thus adequately support LAB viability and enhance stability during freezing and freeze-drying processes and could potentially be better than the conventional MRS medium.

      CONCLUSIONS

      In the present study, a modified growth medium was developed using alternative nitrogen sources (Ohly yeast extracts) to evaluate the fermentation capability of some strains of Lb. bulgaricus. Our results demonstrated that the nitrogen sources N-Max, KAT, and C-Max can efficiently support the growth and viability of Lactobacillus delbrueckii ssp. bulgaricus and could thus be used for the fermentation of LAB or probiotic cultures. In addition, these nitrogen sources could potentially act as cryoprotectants to ensure the freeze-stability and viability of freeze-dried probiotic cells. The AA l-histidine could also be supplemented in growth medium to enhance the medium's buffering capacity. Consequently, N-Max, KAT, and C-Max yeast extracts are superior nitrogen sources that have promising potential for bioprocessing operations and could be employed for dairy starter fermentation processes due to their enhanced growth-promoting factors.

      ACKNOWLEDGMENTS

      This publication was made possible by grant number NC.X-267-5-12-170-1 from the National Institute of Food and Agriculture (NIFA) and in part by NIZO Food Research BV (Ede, the Netherlands), Ohly GmbH (Hamburg, Germany), THT Prayon (Gembloux, Belgium), Jarrow Formulas (Los Angeles, CA), and the Department of Family and Consumer Sciences and the Agriculture Research Station at North Carolina Agriculture and Technical State University (Greensboro, NC). The authors have not stated any conflicts of interest.