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Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10-2 to 10-5. The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase 1.3 gene, which may be involved in the MMEJ event, and the frequency of MMEJ at 4.3 is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ1.3 increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1.3 can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.

In this study, a small-scale simulated fermentation system of fermented grains (Jiupei in Chinese) without pit mud (PM) was used as a control, and the differences in the microbial community and metabolite components between the PM and control groups were compared. The results indicated that PM significantly enhanced the contents of major flavor compounds in Jiupei including butyric acid, caproic acid, ethyl acetate, ethyl butyrate, and ethyl caproate, and reduced the contents of lactic acid and ethyl lactate. For both groups, the relative abundance of Lactobacillus, one of the dominant bacteria in the upper and bottom layers of Jiupei, significantly increased at the initial stage, and showed a slight difference between them at the end of fermentation. The decay period of Lactobacillus in the PM group was about 15 days earlier than that in the control group. At the same time, the relative abundance of Rhodococcus in the control group, and the relative abundance of Kroppenstedtia, Clostridium_sensu_stricto_12, and Acetobacter in the PM group increased. The abundance of most of the enzymes in the Embden-Meyerhof-Parnas (EMP) pathway, as well as that of caproic acid synthase (EC 1.3.1.38, EC 2.3.1.16, EC 6.2.1.1) and butyric acid synthase (EC 2.3.1.9, EC 2.8.3.8) increased during fermentation, and the abundance of these enzymes was significantly higher in the bottom layer of Jiupei than in the upper layer. The abundance of lactate dehydrogenase (EC 1.1.1.27), which uses lactic acid as a substrate, increased in the PM group. These findings reveal the contribution of PM to the microbial communities and metabolite components of Jiupei.

Gas chromatography-mass spectrometry (GC-MS) and electronic tongue were used to quantitatively determine the volatile compounds and taste indices of 21 Jiang-flavor baijiu samples of different grades. These samples were differentiated by chemometrics, and key differential compounds among grades were identified. Finally, a discriminant model was established by machine learning. The results showed that there were differences in the contents of volatile compounds in Jiang-flavor baijiu of three grades, indicating the feasibility of further discriminant analysis. The total content of flavor compounds in second-grade baijiu (4 908 mg/L) was significantly lower than that in premium-grade (6 583 mg/L) and first-grade baijiu (8 254 mg/L), while the proportion of several esters responsible for floral and fruity aromas in total esters showed a decreasing trend as the grade decreased. Partial least squares-discriminant analysis (PLS-DA) identified 16 key differential compounds represented by ethyl palmitate and acetic acid. The results of electronic tongue showed that the taste indexes of premium-grade baijiu were more consistent, with lower bitterness and astringency aftertaste. The taste indexes of second-grade baijiu showed significant intersample differences. Principal component analysis (PCA) showed clear discrimination of Jiang-flavor baijiu of different grades according to their taste indexes. The above results provide a basis for the establishment of Jiang-flavor baijiu quality system. Four discriminant models were established based on 25 differential compounds and taste indexes identified. The accuracy of all models was higher than 90%, and the support vector machine (SVM) model performed best, with an accuracy of 100%.

The effect of spatial heterogeneity on the microbial community and physicochemical properties during the primary fermentation of Daqu were investigated by high-throughput sequencing technology and conventional detection methods. Nongxiangxing baijiu Daqu inoculated with the unique ripe starter obtained by gradually culturing and expanding Daqu treated by cosmic rays was used. The results showed that although the intensity of change in driving factors varied among layer, their trends were the same. The liquefying, saccharifying and esterifying power of Daqu were higher in the bottom layer than in the upper and middle layers at the same fermentation time and the fluctuation was small. The microbial community of Daqu was composed of 12 dominant bacterial genera, including Lactobacillus, Weissella, Bacillus, Kosakonia, Staphylococcu and Thermoactinomyces, and seven dominant fungal genera, such as Pichia, Thermoascus, and Rhizomucor. Principal co-ordinates analysis and hierarchical clustering analysis showed significant differences in the bacterial and fungal community structure among fermentation stages and layers. Procrustes analysis and Mantel test showed that moisture had a significant effect on the bacterial community in Daqu, and acidity had a significant effect on the bacterial community in the middle and bottom layers of Daqu. Moreover, moisture had a significant effect on the fungal community in the upper and middle layers of Daqu. Redundancy analysis showed that moisture and acidity were positively correlated with Lactobacillus and Pichia, while driving factors had different influences on the microbial communities in different layers of Daqu. Therefore, the interaction and co-occurrence patterns of microbial genera in Daqu could change due to the differences in driving factors among different layers of Daqu. These results suggested that regulating driving factors during the Daqu making process is an effective way to improve the microbial community structure and quality of Daqu.

Mulberry perishes easily due to its high water content and thin skin. It is important to extend the shelf life of mulberry by proper processing methods. In the present study, the influence of three drying techniques, including hot air drying (HAD), vacuum drying (VD), and vacuum freeze-drying (VFD) on the quality maintenance of mulberry was comprehensively evaluated. Bioactive compounds, antioxidant activity, and the sensory and volatile flavor compounds of mulberry have been researched. The results showed that VFD treatment maintained the highest anthocyanins (6.99 mg/g), total flavones (3.18 mg/g), and soluble sugars (2.94 mg/g), and exhibited the best DPPH· (81.2%) and ABTS+· (79.9%) scavenging ability. Mulberry also presented the lowest hardness and the greatest brittleness after VFD. Additionally, VFD maintained the optimal color and presented the best sensory attributes. Furthermore, 30, 20, and 32 kinds of volatile flavor compounds were detected in HAD, VD, and VFD, respectively, among which aldehydes, esters, and ketones were the most abundant compounds. This study indicated the potential application value of VFD for the drying of fruit and vegetable foodstuffs. [ABSTRACT FROM AUTHOR]