Fermentation Profile and Probiotic-Related Characteristics Of

fermentation Article Fermentation Profile and Probiotic-Related Characteristics of Bifidobacterium longum MC-42 Anna V. Begunova 1, Irina V. Rozhkova 1, Olga A. Glazunova 2,* , Konstantin V. Moiseenko 2, Olga S. Savinova 2 and Tatyana V. Fedorova 2,* 1 Federal State Budgetary Scientific Institution “All-Russian Research Institute of Dairy Industry”, 115093 Moscow, Russia; [email protected] (A.V.B.); [email protected] (I.V.R.) 2 Research Center of Biotechnology of the Russian Academy of Sciences, A.N. Bach Institute of Biochemistry, 119071 Moscow, Russia; [email protected] (K.V.M.); [email protected] (O.S.S.) * Correspondence: [email protected] (O.A.G.); [email protected] (T.V.F.) Abstract: This article presents new data on Bifidobacterium longum MC-42—a strain that has been actively used for the preparation of commercial dairy products in Russia for almost 40 years. It was demonstrated that this strain possesses high activities of b-galactosidase, α-glucosidase, and leucine arylaminidase; inhibits the growth of pathogens such as Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli; and can efficiently remove cholesterol from the cultural medium. The resistance of B. longum MC-42 determined for 15 commonly used antibiotics was in agreement with those previously reported for Bifidobacterium spp. The absence of frequently transmittable antibiotic resistance genes in the genome and the lack of undesirable activity of b-glucuronidase proved the safe use of B. longum MC-42 as a probiotic and starter culture. Additionally, the impact of two growth-promoting additives—yeast extract or milk protein hydrolysate containing supplementation— Citation: Begunova, A.V.; Rozhkova, I.V.; Glazunova, O.A.; Moiseenko, on the B. longum MC-42 fermentation profile was assessed. The introduction of these additives K.V.; Savinova, O.S.; Fedorova, T.V. increases the maximum attainable viable cell count by orders of magnitude, significantly changed Fermentation Profile and the profile of aminopeptidase activities in extracellular extracts, and influenced the antioxidant and Probiotic-Related Characteristics of antihypertensive properties of the obtained fermented products. Bifidobacterium longum MC-42. Fermentation 2021, 7, 101. Keywords: bifidobacteria; Bifidobacterium longum; aminopeptidase activity; antioxidant activity; https://doi.org/10.3390/ angiotensin-I-inhibitory activity; cholesterol removal capacity; antibacterial activity fermentation7030101 Academic Editor: Hiroshi Kitagaki 1. Introduction Received: 26 May 2021 In recent years, probiotics—living microorganisms providing health benefits upon Accepted: 21 June 2021 Published: 26 June 2021 adequate consumption—have gained incredible attention both from ordinary customers and professional scientists [1]. While it was originally thought that probiotics promote Publisher’s Note: MDPI stays neutral physical well-being primarily through the improvement of microbial balance, later research with regard to jurisdictional claims in demonstrated that the action of probiotics on organism goes far beyond direct interaction published maps and institutional affil- with a consumer’s indigenous microflora [2]. The number of recently discovered properties iations. of probiotics, often with strain-specificity such as improvement of intestinal barrier [3], production of beneficial enzymes (e.g., b-galactosidase and bile salt hydrolase) [4], modula- tion of immune system [5], synthesis of neurochemicals [6], and production of bioactive peptides [7], stimulates the search for new probiotic strains and reevaluation of old ones. Bifidobacterium is a widely known genus of probiotic bacteria [8]. Being first introduced Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. with breast milk, bifidobacteria constitutes 60–70% of the fecal bacteria in infancy and This article is an open access article 30–40% in adulthood [9]. The long history of bifidobacteria consumption (both as part of distributed under the terms and fermented milk microflora and as commercially manufactured probiotic products) firmly conditions of the Creative Commons established their safety and positive health benefits [10]. Currently, various strains of Attribution (CC BY) license (https:// Bifidobacterium spp. are used in commercial products throughout the world. Although creativecommons.org/licenses/by/ almost all of these strains were isolated many decades ago, an investigation on their 4.0/). beneficial properties are still actively carried out. Recently, systematical reviews regarding Fermentation 2021, 7, 101. https://doi.org/10.3390/fermentation7030101 https://www.mdpi.com/journal/fermentation Fermentation 2021, 7, 101 2 of 13 properties of several commercialized bifidobacterial strains, such as Bifidobacterium animalis subsp. lactis BB-12 [11], Bifidobacterium bifidum BGN4 [12], and Bifidobacterium breve M- 16V [13], were published in scientific literature. The strain Bifidobacterium longum MC-42 was isolated from the feces of infants by the staff of Scientific Research Institute of the Dairy Industry and patented in 1982 (USSR patent № 863639); at the time of isolation, it was identified as Bifidobacterium adolescentis. In the past 40 years, this strain was actively used (both alone and as part of a mixed starter culture) for the preparation of commercial dairy products and, currently, can be purchased in lyophilized form as a dietary supplement. Although advantageous technological properties of this strain such as the high viability of a lyophilized culture and the good sensory properties of the fermented products were time-tested, many aspects of its probiotic properties remain unclear. Only recently, the genome of B. longum MC-42 was sequenced [14], and the response of a human gut microbiome on short-term supplementation of this strain was investigated [15]. The aim of the current study was to investigate the B. longum MC-42 probiotic properties by evaluation of its profile of enzymatic activities, its ability to inhibit the growth of common pathogens, its resistance to common antibiotics, its possession of transmittable antibiotic resistance genes, and its capacity for cholesterol removal. Additionally, the fermentations of skim milk and skim milk supplemented with two different growth promoting additives (yeast extract and milk protein hydrolysate containing supplementation) performed by B. longum MC-42 were described in terms of strain’s growth characteristics, acidification capability, and proteolytic activity. The resulting fermented products were assessed in vitro for their antioxidant and antihypertensive properties. 2. Materials and Methods 2.1. Strain Reactivation and Preparation of Starting Inoculum The strain B. longum MC-42 was obtained from the Microorganism Collection of the All-Russian Research Institute of the Dairy Industry (VNIMI, Moscow, Russia), where it was stored as a lyophilized culture at −80 ◦C. The strain was reactivated in commercial milk hydrolysate containing corn-lactose GMK-2 medium for bifidobacteria (Biokompas-S, Uglich, Russia) at 37 ◦C for 24–48 h (to achieve a turbidity of 0.5–1 McFarland standard). For all experiments, the starting inoculum was prepared by the addition of 3% (v/v) of a reactivated strain into 200 mL of a GMK-2 medium with subsequent incubation at 37 ◦C for 24–48 h (to achieve a turbidity of 0.5–1 McFarland standard) and adjustment to approximately 107 CFU·mL−1. An evaluation of the enzymatic activities of B. longum MC-42 was carried out using the API ZYM kit (BioMerieux, Marcy-l’Étoile, France), according to the manufacturer instructions. For analysis, B. longum was grown in MRS broth for 72 h at 37 ◦C. 2.2. Assessment of Inhibition of Pathogens The pathogenic bacterium strains Staphylococcus aureus ATCC-6538 and Escherichia coli ATCC-25922 were purchased from American Type Culture Collection (Manassas, VA, USA), and Salmonella typhimurium NCTC 00074 was purchased from National Collection of Type Cultures (Salisbury, UK). The strains were cultivated on agar slants (21.0 g·L−1 peptone, −1 −1 −1 −1 −1 6.5 g·L NaCl, 6.25 g·L glucose, 3.5 g·L Na2HPO4, 0.6 g·L KH2PO4, and 12.5 g·L agar) for 24 h at 37 ± 2 ◦C. For inoculation, the cell suspension was washed off the agar slant and diluted to approximately 107 CFU·mL−1. The antagonistic activity was evaluated by the co-culture method [16]. For the ex- perimental samples, 20 mL of MRS broth was simultaneously inoculated with 1 mL of B. longum MC-42 and 1 mL of the pathogenic test-strain starting inocula; for the control samples, only inoculation with the pathogenic test-strain was performed. The incubation was carried out at 37 ± 2 ◦C, and samples were collected after 24 and 48 h. Pathogen cells were counted at SPA agar medium (Mikrogen, Moscow, Russia) at 37 ◦C for 24–48 h. Fermentation 2021, 7, 101 3 of 13 2.3. Antibiotic Resistance Assays The antibiotic resistance of B. longum MC-42 was assessed by the disk diffusion method. MRS agar plates were inoculated with 1 mL of the B. longum MC-42 starting inoculum, and antibiotic disks (DI-PLS-50-01, NICF, St. Peterburg, Russia) were placed in the center of each plate. The plates were incubated under anaerobic conditions using Oxoid AnaeroJar 2.5 L (Thermo Fisher Scientific, USA) and GasPak sachets (BD Biosciences, San Jose, CA, USA) for 18–24 h at 37 ◦C, followed by measurement of the inhibition zone diameters. 2.4. Search for Genes of Interest in Genome To search for genes of interest, the genome of B. longum MC-42 was downloaded from the NCBI GenBank database

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