Citation: Kiš, M.; Zdolec, N.; Kazazi´ c, S.; Vincekovi´ c, M.; Juri´ c, S.; Dobrani´ c, V.; Oštari´ c, F.; Mari´ c, I.; Mikulec, N. Implementation of Novel Autochthonous Microencapsulated Strains of Lactiplantibacillus plantarum, Lactococcus lactis, and Lamb’s Rennet in the Production of Traditional “Paški Sir” Cheese. Fermentation 2023, 9, 441. https://doi.org/10.3390/ fermentation9050441 Academic Editor: Thomas Bintsis Received: 20 April 2023 Revised: 28 April 2023 Accepted: 3 May 2023 Published: 5 May 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/). fermentation Article Implementation of Novel Autochthonous Microencapsulated Strains of Lactiplantibacillus plantarum, Lactococcus lactis, and Lamb’s Rennet in the Production of Traditional “Paški Sir” Cheese Marta Kiš 1 , Nevijo Zdolec 1, * , Snježana Kazazi´ c 2 , Marko Vincekovi´ c 3 , Slaven Juri´ c 3 , Vesna Dobrani´ c 1 , Fabijan Oštari´ c 3 , Ivan Mari´ c 2 and Nataša Mikulec 3 1 Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia; mkis@vef.hr (M.K.); vdobranic@vef.hr (V.D.) 2 Ru ¯ der Boškovi´ c Institute, 10000 Zagreb, Croatia; snjezana.kazazic@irb.hr (S.K.); imaric@irb.hr (I.M.) 3 Faculty of Agriculture, University of Zagreb, 10000 Zagreb, Croatia; mvincekovic@agr.hr (M.V.); sjuric@agr.hr (S.J.);fostaric@agr.hr (F.O.); nmikulec@agr.hr (N.M.) * Correspondence: nzdolec@vef.hr Abstract: This study aimed to implement a microencapsulated form of selected autochthonous lactic-acid bacteria (LAB) isolated from the cheese-production chain and natural rennet obtained from suckling lambs in the traditional production of hard sheep cheese, “Paški sir”, from the island of Pag, Croatia. Two different formulations of microparticles were prepared: (i) microparticles containing the strain of both Lactiplantibacillus plantarum and Lactococcus lactis (S2) and (ii) microparticles containing both strains and natural rennet (S3). These formulations were used in the production of Paški sir cheese simultaneously with standard production using non-encapsulated commercial starter cultures and commercial rennet (S1). The number of Lc. lactis isolates decreased at day 30 and were not isolated during the remaining ripening process, whereas the number of L. plantarum remained stable throughout the ripening process. The level of LAB and the release of the rennet from microsphere formulations at the end allowed for the production of cheese with the same characteristics as the commercial product, indicating no negative interactions of natural rennet, bacterial culture, and chemical components of microparticles. To our knowledge, this is the first report of a microencapsulated L. plantarum (isolated from the abomasum of lambs) coupled with natural lamb’s rennet used in the production of hard sheep cheese. This pilot study showed the great potential for maintaining authenticity in cheese production by combining traditional and sustainable innovative technologies. Keywords: microencapsulation; Lactiplantibacillus plantarum; Lactococcus lactis; natural rennet; lamb abomasum; Paški sir 1. Introduction The development and introduction of novel technologies in the food industry have always been a priority to improve existing production and food quality. An example of such a technological process is microencapsulation, in which active ingredients are encap- sulated in a suitable polymer material to obtain microparticles capable of retaining the biological activity of the encapsulated ingredient. In the food industry, using microencap- sulation to protect, isolate, or control the release of an active ingredient offers numerous advantages [1–3] and mainly refers to the use of bioactive molecules and living cells [4]. In the dairy industry, especially in cheese production, the potential is closely related to the encapsulation of starter cultures [5–7], which may improve conditions during the ripening process compared to traditional inoculation. Moreover, the added value of such products lies in the bioactive compounds isolated from various natural sources, as they contribute to Fermentation 2023, 9, 441. https://doi.org/10.3390/fermentation9050441 https://www.mdpi.com/journal/fermentation