Citation: Nogacka, A.M.; Cuesta, I.; Gueimonde, M.; de los Reyes-Gavilán, C.G. 2-Fucosyllactose Metabolism by Bifidobacteria Promotes Lactobacilli Growth in Co-Culture. Microorganisms 2023, 11, 2659. https://doi.org/10.3390/ microorganisms11112659 Academic Editors: Xiaoxi Xu and Yanfeng Tuo Received: 11 October 2023 Revised: 25 October 2023 Accepted: 27 October 2023 Published: 29 October 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Article 2-Fucosyllactose Metabolism by Bifidobacteria Promotes Lactobacilli Growth in Co-Culture Alicja M. Nogacka 1,2, * , Isabel Cuesta 1 , Miguel Gueimonde 1,2 and Clara G. de los Reyes-Gavilán 1,2 1 Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), 33300 Villaviciosa, Asturias, Spain; icuesta@ipla.csic.es (I.C.); mgueimonde@ipla.csic.es (M.G.); greyes_gavilan@ipla.csic.es (C.G.d.l.R.-G.) 2 Institute of Health Research of the Principality of Asturias (ISPA), 33011 Oviedo, Asturias, Spain * Correspondence: alicja.nogacka@ipla.csic.es Abstract: Breastfeeding is recognized as the gold standard in infant nutrition, not only because of breastmilk’s intrinsic nutritional benefits but also due to the high content of different bioactive com- ponents such as 2-fucosyllactose (2 0 FL) in the mother’s milk. It promotes the growth of its two major consumers, Bifidobacterium longum ssp. infantis and Bifidobacterium bifidum, but the effect on other intestinal microorganisms of infant microbiota remains incompletely understood. pH-uncontrolled fecal cultures from infants donors identified as “fast 2 0 FL -degrader” microbiota phenotype were used for the isolation of 2 0 FL-associated microorganisms. The use of specific selective agents allowed the successful isolation of B. bifidum IPLA20048 and of Lactobacillus gasseri IPLA20136. The charac- terization of 2 0 FL consumption and its moieties has revealed more pronounced growth, pH drop, and lactic acid production after 2 0 FL consumption when both microorganisms were grown together. The results point to an association between B. bifidum IPLA20048 and L. gasseri IPLA20136 in which L. gasseri is able to use the galactose from the lactose moiety after the hydrolysis of 2 0 FL by B. bifidum. The additional screening of two groups of bifidobacteria (n = 38), fast and slow degraders of 2 0 FL, in co-culture with lactobacilli confirmed a potential cross-feeding mechanism based on degradation products released from bifidobacterial 2 0 FL break-down. Our work suggests that this phenomenon may be widespread among lactobacilli and bifidobacteria in the infant gut. More investigation is needed to decipher how the ability to degrade 2 0 FL and other human milk oligosaccharides could influence the microbiota establishment in neonates and the evolution of the microbiota in adult life. Keywords: 2 0 FL; bifidobacteria; lactobacilli; degradation status; cross-feeding 1. Introduction Breastfeeding is recognized as the gold standard in infant nutrition. Its exclusivity is the recommended feed habit for the infant during the first six months of life, with the continuation of breastfeeding up to 2 years of age [1]. This is not only because of breast- milk’s intrinsic nutritional benefits but is also due to the high content of different bioactive components in the mother’s milk. Some of them are the human milk oligosaccharides (HMOs) which represent the third-largest solid component of milk, after lactose and lipids, with a concentration ranging from 9 to 24 g/L. This group of carbohydrates is composed of about 200 different structures formed by fucosylated, sialylated, and neutral oligosaccha- rides with a lactose core at the reducing end, which vary in composition and concentration during lactation [2]. Additional variation in HMO structure can be largely linked to the maternal blood group, by the expression of Secretor and Lewis genes, encoding α1–2 fucosyltransferase (FUT2) and α1–4 fucosyltransferase (FUT3) enzymes, respectively [3]. The functional allele of FUT2 is used to classify mothers as “secretors”, and they produce 2-fucosyllactose (2 0 FL) as the most abundant HMO in milk. In contrast, “non-secretor” mothers produce mainly oligosaccharides with α-1,4- and α-1,3-fucosyl bonds [3]. Given that most women (about 75%) present a “secretor” phenotype [4], 2 0 FL may be regarded as Microorganisms 2023, 11, 2659. https://doi.org/10.3390/microorganisms11112659 https://www.mdpi.com/journal/microorganisms