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11 pages, 2923 KB

Open AccessArticle

Enhancing Protease and Amylase Activities in Bacillus licheniformis XS-4 for Traditional Soy Sauce Fermentation Using ARTP Mutagenesis

by Andong Zhang, Yudong Ma, Yue Deng, Zhiwei Zhou, Yue Cao, Bin Yang, Jing Bai and Qun Sun

Foods 2023, 12(12), 2381; https://doi.org/10.3390/foods12122381 - 15 Jun 2023

Cited by 23 | Viewed by 4021

Abstract

This study was conducted to increase the enzymatic activity of Bacillus licheniformis XS-4, which was isolated from the traditional fermented mash of Xianshi soy sauce. The mutation was induced by atmospheric and room-temperature plasma (ARTP), and a mutant strain, mut80, was obtained. mut80 exhibited significant increases in protease and amylase activity by 90.54% and 143.10%, respectively, and the enhanced enzymatic activities were stably maintained after 20 consecutive incubations. Re-sequencing analysis of mut80 revealed that the mutation sites were located in 1518447(AT-T) and 4253106(G-A) in its genome, which was involved in the metabolic pathways of amino acids. The expression of the protease synthetic gene (aprX) increased 1.54 times, while that of the amylase gene (amyA) increased 11.26 times, as confirmed via RT-qPCR. Using ARTP mutagenesis, the present study proposes a highly efficient microbial resource with enhanced protease and amylase activity provided by B. licheniformis, which can potentially be used to improve the efficiency of traditional soy sauce fermentation. Full article

(This article belongs to the Special Issue Biotechnological Potential of Microorganisms in Food Industry)

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17 pages, 3559 KB

Open AccessArticle

The Impact of Low-Temperature Inactivation of Protease AprX from Pseudomonas on Its Proteolytic Capacity and Specificity: A Peptidomic Study

by Chunyue Zhang, Sjef Boeren, Liming Zhao, Etske Bijl and Kasper Hettinga

Dairy 2023, 4(1), 150-166; https://doi.org/10.3390/dairy4010011 - 3 Feb 2023

Cited by 4 | Viewed by 3571

Abstract

The destabilization of UHT milk during its shelf life can be promoted by the residual proteolytic activity attributed to the protease AprX from Pseudomonas. To better understand the hydrolysis patterns of AprX, and to evaluate the feasibility of using low-temperature inactivation (LTI) for AprX, the release of peptides through AprX activity on milk proteins was examined using an LC-MS/MS-based peptidomic analysis. Milk samples were either directly incubated to be hydrolyzed by AprX, or preheated under LTI conditions (60 °C for 15 min) and then incubated. Peptides and parent proteins (the proteins from which the peptides originated) were identified and quantified. The peptides were mapped and the cleavage frequency of amino acids in the P1/P1′ positions was analyzed, after which the influence of LTI and the potential bitterness of the formed peptides were determined. Our results showed that a total of 2488 peptides were identified from 48 parent proteins, with the most abundant peptides originating from κ-casein and β-casein. AprX may also non-specifically hydrolyze other proteins in milk. Except for decreasing the bitterness potential in skim UHT milk, LTI did not significantly reduce the AprX-induced hydrolysis of milk proteins. Therefore, the inactivation of AprX by LTI may not be feasible in UHT milk production. Full article

(This article belongs to the Special Issue Omics Research in Dairy Sciences)

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15 pages, 1817 KB

Open AccessArticle

Peptidomic Fingerprints of Stored UHT Milk Inoculated with Protease Extracts from Different Pseudomonas Strains Relative to aprX Expression and Visible Spoilage

by Miguel Aguilera-Toro, Søren Drud-Heydary Nielsen, Martin Laage Kragh, Yinghua Xiao, Lisbeth Truelstrup Hansen, Valentin Rauh, Lars Wiking, Nina Aagaard Poulsen and Lotte Bach Larsen

Dairy 2023, 4(1), 83-97; https://doi.org/10.3390/dairy4010005 - 9 Jan 2023

Cited by 6 | Viewed by 4240

Abstract

Lately, concern about the protease AprX produced by Pseudomonas has increased in the dairy industry due to its ability to survive UHT treatment and spoil UHT milk. Efficient prediction methods for UHT milk spoilage are currently lacking, mainly due to high diversity in proteolytic potential between Pseudomonas strains. The present study aimed to gain more insight into the variability between Pseudomonas strains regarding proteolytic potential by comparing their proteolytic capability with their aprX expression levels and differences in peptide formation. The variability in aprX expression levels in four Pseudomonas strains were related to physical stability, milk proteolysis and peptidomic cleavage patterns of milk proteins in a storage experiment of UHT milk inoculated with protease extracellular extracts and stored for 45 days at 20 °C. A positive relationship was observed between the relative expression of aprX and milk proteolysis during storage, with the strain Pseudomonas panacis DSM 18529 showing the highest level in both parameters. This strain was the only strain to show visual gelation, which occurred after 21 days. The peptide formation analysis showed a similar protein hydrolysis pattern between strains and high hydrolysis of α_s1-caseins during long-term spoilage putatively due to the activity of AprX was observed. Full article

(This article belongs to the Special Issue Thermal Processing of Milk and Milk Products)

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14 pages, 1776 KB

Open AccessArticle

Proteolytic Traits of Psychrotrophic Bacteria Potentially Causative of Sterilized Milk Instability: Genotypic, Phenotypic and Peptidomic Insight

by Stefano Morandi, Valentina Pica, Fabio Masotti, Stefano Cattaneo, Milena Brasca, Ivano De Noni and Tiziana Silvetti

Foods 2021, 10(5), 934; https://doi.org/10.3390/foods10050934 - 24 Apr 2021

Cited by 14 | Viewed by 4629

Abstract

The proteolytic traits of the psychrotrophic strains Pseudomonas poae LP5, Pseudomonas fluorescens LPF3, Chryseobacterium joostei LPR1, Pseudomonas fulva PS1, Citrobacter freundii PS37, Hafnia alvei PS46, and Serratia marcescens PS92 were initially investigated by phenotypic and genotypic approaches. Six strains elicited extracellular proteolytic activity, and five expressed the thermostable AprX or (likely) Ser1 enzymes. Then, the strains were inoculated (10⁴ CFU/mL) in microfiltered pasteurized milk and kept at 4 °C for five days. All of the strains reached 10⁸ CFU/mL at the end of storage and five produced thermostable extracellular proteolytic enzymes. The freshly inoculated samples and the corresponding samples at 10⁸ CFU/mL were batch-sterilized (131 °C, 30 s) and kept at 45 °C up to 100 days. The former samples did not gel until the end of incubation, whereas the latter, containing P. poae, P. fluorescens, C. joostei, C. freundii, and S. marcescens, gelled within a few days of incubation. The thermostable proteolytic activity of strains affected the peptidomic profile, and specific proteolyzed zones of β-CN were recognized in the gelled samples. Overall, the results confirm some proteolytic traits of psychrotrophic Pseudomonas spp. strains and provide additional insights on the proteolytic activity of psychrotrophic bacteria potentially responsible for sterilized milk destabilization. Full article

(This article belongs to the Special Issue Dairy Spoilage Microorganisms, Mechanisms and Novel Preservation Strategies)

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