Abstract:In the past few centuries, fermentation engineering has experienced the conversion from natural fermentation into metabolic engineering. The fermentation products were also shifted from food and other means of life to more basic industrial materials. Has made great progress in strain selection, process control, separation technology, etc. In the past three decades, the rapid development of biotechnology has provided an important foundation for the development of fermentation engineering in the new era, enabling scientists and engineers to achieve more goals through fermentation engineering, and supporting other industries, such as pharmaceutical industry, food industry, agriculture and animal husbandry, energy and materials, textile, paper and leather industry, daily chemical industry, environment and ecology. In the 21st century, the contradiction between human sustainable development and ecological environment, resources and energy is increasingly prominent. How to play the key role of fermentation engineering is also a major task of fermentation engineering researchers. The developments of systems biology, synthetic biology, bioinformatics, advanced materials science and so on have also significantly revolutionized fermentation engineering. The utilization of advanced biotechnology can achieve a series of unprecedented changes, such as the transformation of aerobic fermentation to anaerobic fermentation, the artificial synthesis of microbial community of fermented food, the construction of autotrophic microbial fermentation plant, artificial synthetase, etc., so that more difficult challenges in the future could be successfully accomplished by fermentation engineering.

Abstract:Milk allergy is a serious public health problem. In order to reduce the proportion of people suffering from milk allergies, effective prevention and treatment methods need to be sought. The anti-allergic mechanism of lactic acid bacteria is summarized from two aspects. The protease produced by lactic acid bacteria could destroy the epitope of milk allergen. The fermentation of lactic acid bacteria could regulate the immune system balance. The potential and application prospect of lactic acid bacteria in the prevention and treatment of allergic diseases are also introduced in this review.

Abstract:L-arabinose is a unique five-carbon aldose with good physicochemical properties and physiological activities, widely applied in various fields such as food, chemical and pharmaceutical industries. It can inhibit sucrose absorption, improve insulin resistance, reduce serum triglyceride, reduce fat production, and improve intestinal environment. The recent research progress and mechanism of L-arabinose-related physiological activities were summarized in this paper, providing a basis for the further development and utilization of L-arabinose.

Abstract:Bee bread, a fermented mixture of plant pollen, honey and bee saliva, is a natural and nutritious food. A total of 61 strains of Lactobacillus were isolated from the bee bread and identified as Lactobacillus kunkeei by 16S rRNA gene sequencing. The activity of 61 strains of L. kunkeei to degradate cholesterol was detected by ortho-phthalaldehyde (OPA) method. The degradation rates of 5 strains among them were higher than 15%, and the cholesterol degradation rate of B35 reached (29.07±1.30)%. The 5 L. kunkeei strains were further studied to investigate their probiotic characteristics including acid resistance, bile salt tolerance, antibiotic resistance and bacteriostatic activities. Their acid and bile salt resistance tests showed that 5 strains of L. kunkeei had good acid and bile salt tolerance. The antibiotic susceptibility test results showed that the 5 L. kunkeei strains were sensitive to four clinically significant antibiotics including erythromycin, kanamycin, norfloxacin and vancomycin. The antibacterial activity to gram-positive and gram-negative of 5 L. kunkeei strains was tested by agar diffusion method. All the 5 strains inhibited the growth of Escherichia coli and Staphylococcus aureus. The adhesion rate of strain B35 to Caco-2 cells in vitro was 3.32%. In this study, 5 L. kunkeei strains isolated from bee bread showed good probiotic performance, of which strain B35 had the best probiotic properties. This study offers new bacteria sources for the development and utilization of probiotics.

Abstract:Lactobacillus panis has been identified as the major lactic acid-producing bacterium in the fermentation of Maotai-flavor liquor. However, the effects of different environmental conditions on lactic acid metabolism and expression level of key genes in the lactic acid pathways of L. panis have not been studied. In this work, different environmental factors relevant to fermentation processes were selected, such as temperature, lactic acid, ethanol, and glucose concentrations. Results showed that under different environmental conditions, the growth and lactic acid yield of L. panis were significantly regulated. Lactic acid and ethanol were identified to be the key factors of lactic acid biosynthesis by L. panis. The maximum amount (12.3 g/L) of lactic acid was achieved when 10 g/L lactic acid was added to the growth medium. In contrast, the addition of 40 g/L ethanol reduced lactic acid production to 7.2 g/L, which was the lowest level among the different environmental conditions. Quantitative real-time PCR analysis of key genes involved in lactic acid metabolism with lactic acid supplementation showed that lactic acid addition significantly facilitated up-regulation of key gene for lactic acid synthesis, including ldhL and ldhD encoding L-lactic acid dehydrogenase and D-lactic acid dehydrogenase, respectively. However, their expression levels were down-regulated if ethanol added, implying a decreased metabolic flux through the lactic acid synthetic pathway. The analysis of key environmental factors determining lactic acid production of L. panis is of great significance for modulation of lactic acid synthesis during fermentation, and would also be useful for controlling the brewing microbial community and quality of the liquor.

Abstract:The changes of molecular weight parameters of alginates hydrolyzed by alginate lyase (ALyase) and cellulase using one-step and step-by-step methods were studied by GPC-MALLS. The chain shear models and the degradation conditions were determined. The results showed cellulase had no function on alginate if used alone. Multiple shear model and central shear model were observed when alginate lyase used. Furthermore, the utilization rate was improved with the step-by-step method of alginate lyase. The duration of one-step and step-by-step degradation using 2.56~10.24 U/mg alginate lyase were with in 2 h and 5 h, respectively. The synergistic effect of alginate lyase with cellulase was further explored by step-by-step method at 0 min (iACS), 30 min (iACM), 60 min (iACE). Alginate lyase preferred to take effect on the alginates of molecular weight (Mw)≥1×105, while cellulase preferred to 1×104~1×105 g/mol. The PDI values were stable and approached to 2 infinitely in the iACE method, showing a random shear model. In the iACS and iACM methods, the hydrolyzed products varied in components with increased molecular weight, and their PDI values rose to 5.14 and 7.02 respectively.

Abstract:To improve the texture of re-sterilized rice dumplings, a novel germinated brown rice dumplings was investigated with a similar quality with the fresh prepared rice dumplings. With defined addition of germinated brown rice, the effects of fresh-keeping treatments, including hydrogen peroxide spray, hydrogen peroxide spray combined with deoxidization package, and hydrogen peroxide spray combined with alcohol sustained-release tablets, on the preservation and sensory properties of the dumplings were evaluated. The results showed that the dumplings prepared with 51% of germinated brown rice had a natural color and relatively intact structure. The three treatments decreased the aerobic plate count of the dumplings below to 10 CFU/g, which significantly inhibited the microorganisms and less impacted on the texture. The rice dumplings exhibited good smell, natural color, and better sensory properties treated with 3% of hydrogen peroxide, which was then preferred as the preservation condition. Processed in this condition and stored at 25℃ for 15 days,the acid value of germinated brown rice dumplings was lower than 3.0 mg/g and the peroxide value mass fraction was less than 0.15%, according with the standard. In addition, the content of γ-aminobutyric acid remained unchanged during storage.

Abstract:In order to optimize the shaping process of vegetable balls, samples were prepared in this experiment using peas and carrots as main raw materials, together with soybean protein isolate and starch. Single-factor experiments were applied to optimize the conditions of steaming, microwave heating and infrared heating. Measured indexes including hardness, springiness, gel strength, electronic tongue, electronic nose and sensory evaluation were analyzed to investigate the effects of four heating methods (steaming, microwave heating, infrared heating and steaming-microwave heating) on the texture and taste quality for vegetable balls under different boiling time. SPSS analysis showed that the boiling time had a significant effect on texture and taste quality of samples (P<0.05). Compared with the single heating methods, steaming-microwave heating (steaming for 10 minutes combined with microwave heating under 280 w for 40 s) was beneficial to the retention of texture and taste flavor characteristics of vegetable balls. After balls boiled for 2 hours, the hardness retention rate reached 66.6%, and the elastic value was above 0.9. The gel strength retention rate was 70.8%, and the salty and umami taste retention rate was over 89.0%. Steaming combined with microwave heating is thus indicated as an ideal processing method to prepare vegetable balls.

Abstract:Emerging of genome-editing tools such as CRISPR/Cas9 enables us to perform gene knockout (KO) in mammalian cells. However, KO of multiple genes in a cell is inefficient and time consuming. Cell fusion, frequently used for hybridoma construction, is a way to fuse two different cells and create a new cell line with hybrid phenotypes. However, the recessive phenotype by gene-KO cannot be obtained by cell fusion, because gene complementation occurs from the other cell to be fused. The purpose of this study is to develop a method to obtain double KO cells using cell fusion combined with CRISPR/Cas9. The process and method are as follows. Without available cell fusion data using HEK 293. We first knocked out PIGA or PIGK which were necessary for GPI biosynthesis, in HEK 293 wild-type cell line to obtain PIGA-KO cell line and PIGK-KO cell line. Then the two cell lines were as optimized as models. By optimizing the fusion condition, the fusion efficiency of HEK 293 cells was greatly improved. Secondly, we combined cell fusion technology with CRISPR/Cas9 technology to achieve the knockout of multiple sugar genes. Under optimized condition, fusion of FUT8-KO cells and ST6GAL1-KO cells was performed. Both FUT8and ST6GAL1 were successfully disrupted after cell fusion. These results suggest a simple and quick method to acquire double KO cells using our developed method combined cell fusion with CRISPR/Cas9.

Abstract:The application of ultrasound-assisted enzymatic extraction of gelatin from chicken lung was explored in this study. The structure of extracted chicken lung gelatin was investigated by ultraviolet spectroscopy, differential scanning calorimetry, circular dichroism, polyacrylamide gel electrophoresis, laser particle size distribution, scanning electron microscopy, etc. Its emulsifying property was also characterized. The yield of ultrasonic assisted enzymatic extraction was 30.08% based on the dry weight of lungs without impurity, which was 3.43 times compared with that of conventional hot water extraction. And the extraction efficiency increased by 2.15 times, while the thermal denaturation temperature slightly decreased to 90.22 ℃. The characteristic absorption peaks of chicken lung gelatin were observed at 229 nm in both extracted methods. The secondary structure of extracted gelatin using ultrasonic-assisted enzymatic extraction was more orderly with lower degree of random curling. The average particle size distribution of gelatin extracted by ultrasound-assisted enzymatic extraction was 396 nm, reduced by 13.7%. The gelatin molecules distributed by ultrasound assisted enzyme were mainly within 45 000 ~ 95 000, which suggested almost completely degradation into smaller molecular subunits. Obvious fragmentation was observed on their surface microstructures. Insignificant difference of emulsification was determined between two extracted gelatin samples. Therefore, ultrasound-assisted enzymatic extraction can significantly improve the extraction rate of gelatin and effectively shorten the production cycle. Chicken lung gelatin can also be used as an emulsifier in the food industry with a potential application prospect in the food field, which can further improve the added value of broiler by-products.

Abstract:The effect of solid bases on de-esterification of soluble soybean polysaccharides(SSPS) was investigated in this paper. A commercial high-methoxy soybean polysaccharide (SSPS) was used as the raw material, and a solid base MgO was used as the catalyst. The effects of reaction temperature, reaction time and catalyst feed ratio on the de-esterification of SSPS were investigated. The de-esterification effects were estimated by the molecular weight, the degree of esterification (DE), and the monosaccharide composition. The results showed that the molecular weight and DE of product decreasedas the reaction temperature increased, while the polydispersity (PDI) gradually increased. When the catalyst amount increased from 1∶100 to 1∶5, DE decreased significantly. However, the changes in the molecular weight and PDI of the product were relatively small. If further increasing the catalyst feed ratio, all indicators changed insignificantly. As the reaction time prolonged, DE of the product further declined and PDI further increased. The degree of esterification decreased more slowly at the laterreaction stage. The results of monosaccharide composition showed that the neutral sugars in side chains were removed most quickly, while the main chain breaks and de-esterification were almost synchronous. Degradation of polyarabinose and polygalactose side chains was slower than that of the main chain. Compared with the bases de-esterification process, the MgO de-esterification process achieved products with higher molecular weight, lower PDI, and better product color if with a similar degree of esterification. The above results indicated that the MgO de-esterification process would have a good application prospect in future SSPS de-esterification.

Abstract:Bacillus subtilis is an important microbial agent during the aerobic composting. To reduce the production cost and improve the quality and applicability of bacteria, the spore production process of Bacillus subtilis was studied. Firstly, the culture medium and environmental conditions of Bacillus subtilis GX2 were optimized using shaking flasks. Cell concentration was further increased by a batch feed optimization method in a 3 L fermenter. Finally, single-factor and orthogonal experiments were applied to optimize the spore-forming process. The results showed that the number of viable cells and spores in the fermentation broth reached 1.57×10¹⁰ CFU/mL and 1.32×10¹⁰CFU/mL, respectively, and the spore rate was 84.1% when rotating under 150 r/min at pH 6.8 and 50 ℃. To conclude, the optimization of fermentation and spore production for Bacillus subtilis GX2 effectively increased the number of viable cells and spores in the fermentation broth, and reduced the cost of microbial agents. This work provides guidance for Bacillus subtilis culture and lays the foundation for subsequent industrial production and application of aerobic composting.

Abstract:Wuyi Rougui tea is a kind of oolong tea made from the fresh leaves of Cinnamomum cassia(Camellia sinensis cv. Rougui), which is a provincial improved tea tree variety in Fujian Province and deeply favored by consumers because of the characterization of 'rock in the throat, flower in the mouth'. Besides fresh tea leaves, the aroma quality of Wuyi Rougui tea depend greatly on the manufacturing process. However, studies on the metabolic mechanism of aliphatic aroma components represented by green leaf volatiles (GLVs) and fruit esters during process have not been reported. Fresh tea leaves, solar-withering leaves and turn-over leaves of Wuyi Rougui tea were used as test samples. The detection of aliphatic aroma components in samples during manufacture was done by headspace solid phase microextraction (HS-SPEM) combined with gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) . The aliphatic aldehydes were significantly reduced during the turn-over process, while a large amount of aliphatic alcohols and esters were formed and accumulated. Among them, leaf alcohol esters, hexenols and hexenyl esters could responded to water loss and mechanical damage, respectively. The key enzyme activities in the LOX-HPL pathway during process was detected by UV/visible spectrophotometry. The results indicated that the activities of lipoxygenase(LOX), alcohol dehydrogenase(ADH) and alcohol acyltransferases (AAT) reached their peak values in the third time(3T), the fourth time(4T) and the fifth time(5T) of turn-over process, respectively. The results of quantitative reverse transcription polymerase chain reaction(qRT-PCR) demonstrated that CsLOX2 and CsLOX5 genes had a higher relative expression levels in the initial stage of turn over (2T, 3T), and the up-regulation of relative expression of CsADH2-1 and CsAAT genes was observed during the late stage of turn over (4T, 5T) when the process advanced. The correlation between aliphatic aroma, enzyme activity and gene expression during manufacturing pocess of Wuyi Rougui tea was evaluated by Pearson coefficient. A large number of aliphatic alcohols with natural floral and fruity flavors such as 1-hexanol, cis-3-hexenyl isovalerate, hexanoic acid, hexyl ester, cis-3-hexenyl-methylbutyrate and cis-3-hexenyl salicylate, had significant(P<0.05) or extremely significant(P<0.01) positive correlation with the CsADH2-1 gene. Meanwhile, CsAAT gene had similar expression pattern with CsLOX2 and CsLOX5 genes during manufacturing process(r=0.762，r=0.732). Co-expression of key genes in the LOX-HPL pathway drove the synthesis and accumulation of aliphatic aromas during manufacture, laying an important basis for the formation of the aroma-related volatiles of Wuyi Rougui tea.