Diversity of bacterial lactase genes in intestinal contents of mice with antibiotics-induced diarrhea

AIM To investigate the diversity of bacterial lactase genes in the intestinal contents of mice with antibiotics-induced diarrhea.METHODS Following 2 d of adaptive feeding, 12 specific pathogenfree Kunming mice were randomly divided into the control group and model group. The mouse model of antibiotics-induced diarrhea was established by gastric perfusion with mixed antibiotics(23.33 m L·kg^(-1)·d^(-1)) composed of gentamicin sulfate and cephradine capsules administered for 5 days, and the control group was treated with an equal amount of sterile water. Contents of the jejunum and ileum were then collected and metagenomic DNA was extracted, after which analysis of bacterial lactase genes using operational taxonomic units(OTUs) was carried outafter amplification and sequencing.RESULTS OTUs were 871 and 963 in the model group and control group, respectively, and 690 of these were identical. There were significant differences in Chao1 and ACE indices between the two groups(P < 0.05). Principal component analysis, principal coordination analysis and nonmetric multidimensional scaling analyses showed that OTUs distribution in the control group was relatively intensive, and differences among individuals were small, while in the model group, they were widely dispersed and more diversified. Bacterial lactase genes from the intestinal contents of the control group were related to Proteobacteria, Actinobacteria, Firmicutes and unclassified bacteria. Of these, Proteobacteria was the most abundant phylum. In contrast, the bacterial population was less diverse and abundant in the model group, as the abundance of Bradyrhizobium sp. BTAi1, Agrobacterium sp. H13-3, Acidovorax sp. KKS102, Azoarcus sp. KH32 C and Aeromonas caviae was lower than that in the control group. In addition, of the known species, the control group and model group had their own unique genera, respectively.CONCLUSION Antibiotics reduce the diversity of bacterial lactase genes in the intestinal contents, decrease the abundance of lactase gene, change the lactase gene strains, and transform their structures.

Thermostable ethanol tolerant xylanase from a cold-adapted marine species Acinetobacter johnsonii

A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.

Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

Fermentation Performance and Characterization of Cold-Adapted Lipase Produced with Pseudomonas Lip35

Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shaking flasks in a fermentation medium in various nutritional and physical environments. Lipase production has been influenced by the presence of yeast-extract, soybean powder, NaCI, and Tween-80. Maximum lipase productivity was obtained when the physical environment of the fermentation medium was optimal for 67 h. The production of lipase reached 58.9 U·mL^-1. The lipase of Pseudomonas Lip35 can be considered to be inducible, but the inducer had little influence on the production of lipase. The lipase was characterized and showed high lipolytic activity from pH 7.5-8.0. The optimum temperature was observed at 20℃ and the thermal inactivation of lipase was obvious at 60℃. The lipase activity was inhibited by K＋, stimulated by Ca^2＋, and thermostability decreased in the presence of Ca^2＋, therefore the lipase was Ca^2＋ -dependent cold-adapted enzyme.

Lactase non-persistence and milk consumption in Estonia

AIM： To define the frequency of the cfr-13910 variant associated with lactase persistence/non-persistence trait and to analyze the milk consumption of lactase non- persistent subjects in Estonia. METHODS： We genotyped 355 Estonians by polymerase chain reaction and direct sequencing, Milk consumption was analyzed by a questionnaire, specially developed to analyze milk consumption and abdominal complaints. RESULTS： The frequency of the genotype of the C/ C-13910 （lactase non-persistence） was found to be 24.8% in native Estonians. No other single nucleotide polymorphisms covering the region of 400 bp adjacent to the C/T-13910 variant were found. Lactase non- persistence subjects were found to consume less milk than lactase persistence subjects. CONCLUSION： The frequency of lactase non- persistence defined by the C/C-13910 genotype confirms the results of the previous studies based on indirect methods of determining hypolactasia, Milk consumption of lactase non-persistence subjects is consistent with previously reported figures of adult-type hypolactasia in Estonia, However, lactase non-persistence does not prevent the intake of milk in many adults.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Dietary Control of Lactase Expression in the Weaning Rat

The decline in lactase activity during weaning has been well established. However, its molecuIar mechanism remains to be explored. We studied changes in the expression of lactase in terms of the transcription and translation processes in rat microvillus membrane by Northern blot and Western blot analysis, respectively. To examine the effect of dietary change from a milk to a non-milk diet on the developmental pattern of lactase expression, weaning was prevented by keeping the rats under suckling conditions for 27 days after birth. This treatment only suppressed the extent of decline: while the weanlings showed 17 percent activity compared to that of 4-day-old rats, the prolonged suckling rats showed only 42 percent. The changes in the expression of lactase mRNA and protein were parallel with the change of lactase activity. In other words, the fundamental pattern of significant depression of lactase expression occurred relatively independent of dietary modification.This observation indicates that the regulation of lactase expression is firmly determined at the transcriptional level, and that dietary factor such as the termination of lactose ingestion has only a relatively minor effect

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride （PMSF） and hydrochloride 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF）, indicating that it was a serine protease. The presence of Cae＋ and Mnz＋ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid （AA） residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

Molecularly defined adult-type hypolactasia among working age people with reference to milk consumption and gastrointestinal symptoms

AIM: To study milk consumption and subjective milk- related symptoms in adults genotyped for adult-type hypolactasia. METHODS: A total of 1900 Finnish adults were genotyped for the C/T-13910 variant of adult-type hypolactasia and filled in a structured questionnaire concerning milk consumption and gastrointestinal problems. RESULTS: The C/C-13910 genotype of adult-type hypolactasia was present in 18% of the study population. The prevalence of the C/C-13910 genotype was higher among subjects who were undergoing investigations because of abdominal symptoms (24%, P < 0.05). Those with the C/C-13910 genotype drank less milk than subjects with either the C/T-13910 or the T/T-13910 genotype of lactase persistence (18% vs 38%; 18% vs 36%, P < 0.01). Subjects with the C/C-13910 genotype had experienced more gastrointestinal symptoms (84%) during the preceding three-month period than those with the C/T-13910 (79%, P < 0.05) or the T/T-13910 genotype (78 %, P < 0.05). Only 9% (29/338) of the subjects with the C/C-13910 genotype consumed milk and reported no symptoms from it.CONCLUSION: Gastrointestinal symptoms are more common among adults with the C/C-13910 genotype of adult-type hypolactasia than in those with genotypes of lactase persistence.

佛芍颗粒对小鼠肠道菌群及乳糖酶活性的调控作用研究

目的明确佛芍颗粒对小鼠肠道菌群失调及乳糖酶活性的调节作用,为临床应用提供基础研究数据支持。方法实验小鼠按体质量随机分为正常组,模型组,吗丁啉组(12 mg·kg^(-1)),四逆散组(3 g·kg^(-1)),佛芍颗粒高剂量组(48 g生药·kg^(-1))、佛芍颗粒中剂量组(24 g生药·kg^(-1))、佛芍颗粒低剂量组(12 g生药·kg^(-1)),共7组,每组10只,雌雄各半。采用头孢拉定胶囊和硫酸庆大霉素(2∶1)混合物灌胃诱导肠道菌群失调模型小鼠,通过检测小鼠肠道总厌氧菌、乳酸杆菌、双歧杆菌、大肠杆菌数量及结肠黏膜乳糖酶活性水平,综合评价佛芍颗粒对菌群失调和乳糖酶活性的调控作用。结果佛芍颗粒可以明显增加总厌氧菌(P<0.01)、乳酸杆菌(P<0.01)、双歧杆菌(P<0.01)及大肠杆菌(P<0.01)的数量,并提高结肠黏膜乳糖酶活性。结论佛芍颗粒可以改善头孢拉定胶囊和硫酸庆大霉素诱导的菌群失调和乳糖酶活性降低,为佛芍颗粒的临床应用提供参考。

一种高耐热乳糖酶的异源表达、固定化及酶学性质研究

乳糖酶在乳制品生产中具有重要作用,但是目前商用的乳糖酶耐热性低,可重复利用率差。为了解决以上问题,该实验用枯草芽孢杆菌WB800异源表达了激烈热球菌(Pyrococcus furiosus)来源的β-半乳糖苷酶(β-galactosidase,BgaS)。该酶最适温度为90℃,最适pH值为7,具有良好的应用潜力。为了提高该乳糖酶的重复利用率,该实验用壳聚糖微球为载体,采用交联法实现了BgaS的固定化并对交联条件进行优化,确定最适交联条件为:戊二醛26.50 g/L,交联温度35℃,交联时间2.5 h,酶添加量5.94 U/mL,优化后酶活力回收率达55.11%。固定化酶相较游离酶,最适pH与最适温度相同,且在非最适pH条件下,固定化酶的相对酶活力较游离酶普遍提升了18.23%以上,并且固定化酶在95℃条件下孵育90 min仍保有80%以上的相对活性,较游离酶提升了11.31%。并且固定化酶回收利用率高,操作稳定性好,最适条件下处理3 h即可使牛乳中乳糖含量降低至0.5%以下,达到无乳糖水平,为工业化应用提供了技术支撑。

玉竹水提物对抗生素致菌群失调小鼠肠道乳糖酶活性及菌群多样性的影响

目的本研究拟探究玉竹水提物对菌群失调小鼠的乳糖酶活性和肠道菌群多样性的影响。方法将SPF级雄性BALB/c小鼠随机分为4组,分别为对照组(control)、模型组(model)及玉竹低剂量(yzlow,1.56 g·kg^(-1)·d^(-1))和高剂量(yz-high,3.12 g·kg^(-1)·d^(-1))给药组,每组8只(对照组5只)。除对照组外,采用混合抗生素灌胃造模7天后,给药组连续灌胃玉竹7天,对照组与模型组给予等量无菌水。记录小鼠腹泻情况、体质量、摄食量的变化;检测结肠HE染色切片、结肠组织ZO-1蛋白、IL-6表达情况和血清LPS浓度;收集粪便,采用比色法测定乳糖酶活性,并运用16S rRNA高通量测序技术检测并分析肠道菌群的变化。结果抗生素干预7天后,与对照组相比,模型组小鼠大便稀软,体质量减轻、摄食量减少,肠道菌群多样性明显降低。第14天,模型组结肠可见溃疡伴有间质轻微充血水肿,ZO-1表达显著降低,IL-6表达显著升高,血清LPS显著升高,乳糖酶活性显著降低,肠道菌群多样性仍低于正常组;给药7天,与模型组相比,玉竹水提物降低了小鼠的腹泻率并促使体质量及摄食量回升,下调病理结肠组织损伤,ZO-1蛋白表达显著升高,降低结肠因子IL-6、血清LPS浓度。此外,玉竹水提物可显著上调乳糖酶活性,提高菌群失调小鼠的群落丰富度、多样性,表现为回调了菌群失调小鼠的3种差异菌门(上调厚壁菌门,下调变形菌门和拟杆菌门)与7种差异菌属(包括上调Rikenellaceae_RC9_gut_group属、Rikenella属、Colidextribacter属、norank_f__Lachnospiraceae属、norank_f__Oscillospiraceae属、Lachnospiraceae_NK4A136_group属,下调Alloprevotella属),上述菌丰度均与体质量、乳糖酶活性、血液LPS、结肠炎症因子IL-6呈显著相关。结论玉竹水提物能缓解由抗生素导致菌群失调引起的肠道屏障损伤和功能紊乱,其机制可能是通过调节肠道菌群结构。

低乳糖发酵马乳的制备工艺研究及其对乳糖不耐受模型小鼠的影响

通过单因素实验和响应面实验筛选出低乳糖发酵马乳的处方组成及制备工艺。以琼脂添加量、甜菜纤维添加量、乳糖酶添加量、乳酸菌粉添加量为考察因素,分别以葡萄糖含量、酸度、pH和感官评分为评价指标,通过单因素及响应面法设计Box-Behnken响应面实验,筛选并优化低乳糖发酵马乳的最佳制备工艺参数;进一步对乳糖不耐受模型小鼠用低乳糖发酵马乳干预,ELISA试剂盒分别检测肝脏组织中TNF-α、IL-6、IL-1β、IL-10、DAO、D-乳酸、SOD、MDA等含量,进行动物试验考察。当琼脂添加量为0.4%、甜菜纤维0.3%、乳糖酶0.06%、菌粉1.5%时,产品各项指标最理想,筛选出的制备工艺参数稳定、可靠。使用低乳糖发酵马乳对乳糖不耐受小鼠未见加重症状的趋势,适宜乳糖不耐受人群使用。

低乳糖牛乳粉品质分析及蛋白特性研究

为获得品质优良的低乳糖牛乳粉,该试验以生牛乳为原料,分别使用3种不同乳糖酶水解牛乳,对水解后的牛乳进行喷雾干燥得到了低乳糖牛乳粉-乳糖酶A(low lactose milk powder-enzyme A,LLMP-EA)、低乳糖牛乳粉-乳糖酶B(low lactose milk powder-enzyme B,LLMP-EB)、低乳糖牛乳粉-乳糖酶C(low lactose milk powder-enzyme c,LLMP-EC),测定其理化指标和游离巯基等蛋白特性。结果表明,LLMP-EA、LLMP-EB和LLMP-EC的外观及组织状态相近、色泽偏白,LLMP-EA的水分活度比LLMP-EB、LLMP-EC分别低6.29%、4.07%(P<0.05);LLMP-EA的水分含量比LLMP-EB、LLMP-EC分别低6.13%、3.32%(P<0.05);LLMP-EA多分散性指数(polydispersity index,PDI)值为0.13,LLMP-EA、LLMP-EB和LLMP-EC的Zeta电位无显著性差异(P>0.05);通过扫描电镜(scanning electron microscope,SEM)观察发现,LLMP-EA乳粉颗粒较为饱满、表面光滑、有轻微凹陷、颗粒聚集程度较低;LLMP-EA的游离巯基含量比LLMP-EB、LLMP-EC分别低19.90%、12.15%(P<0.05),而LLMP-EA表面疏水性比LLMP-EB低12.29%(P<0.05);LLMP-EA的α-螺旋结构分别比LLMP-EB、LLMP-EC高1.35%、1.94%(P<0.05)。因此,LLMP-EA的水分含量和水分活度低、PDI值仅为0.13,品质最佳;游离巯基含量和溴酚蓝结合量低、α-螺旋含量高,蛋白特性较好。

特异性乳糖酶的开发研究进展

乳糖酶是一种重要的生物催化剂,基于其水解和转糖苷功能,能水解乳糖和生产低聚半乳糖。在乳糖酶的实际应用中,受复杂反应体系和反应条件(不适宜的酸碱度、高温和有机溶剂)的影响,乳糖酶的稳定性和酶活性较差,制约了乳糖酶的应用。因此,开发适应工业应用条件的特异性乳糖酶及其高效生产的研究一直是研究者们关注的焦点。本文在介绍耐热乳糖酶、低温乳糖酶、耐盐乳糖酶、酸性乳糖酶和耐溶剂化乳糖酶的研究的基础上,综述特异性乳糖酶的催化机制、高产菌株培育、定向进化以及应用策略等方面的进展,并对工业应用前景进行展望,以期为特异性乳糖酶的深入探索和应用开发提供参考。

新生儿乳糖酶缺乏及乳糖不耐受危险因素分析

目的分析新生儿乳糖酶缺乏及乳糖不耐受的危险因素,为临床前瞻性预防乳糖不耐受提供临床依据。方法选取2021年1月-2022年12月常州市儿童医院新生儿科病房住院的259名的新生儿为研究对象,收集胎龄、年龄、体重、Apgar评分、喂养等临床资料,所有患儿均排空尿液并饮奶10ml/kg,留取饮奶后1-2 h期间的尿液10ml,2h内送检,行尿半乳糖测定,检测结果阳性者为乳糖酶缺乏。如果乳糖酶缺乏同时伴有腹胀、腹痛、呕吐等临床症状为乳糖不耐受。根据乳糖酶缺乏与否分为乳糖酶缺乏组及乳糖酶不缺乏组,尿乳糖阳性并存在乳糖不耐受症状的为乳糖不耐受组。比较临床资料的差异,进行单因素分析和Logistic回归分析新生儿乳糖酶缺乏及乳糖不耐受的风险因素。结果①单因素分析及三组间交叉分割,乳糖酶缺乏组和不缺乏组间除了在胎龄25-33周、出生体重两组之间差异有统计学意义(P<0.01)外,在年龄、呼吸支持模式、喂养方式、抗生素应用、激素应用等方面均无显著性差异(P>0.017);乳糖酶缺乏组和乳糖不耐受组间在胎龄、出生体重、年龄等所有研究因素方面均无显著性差异(P>0.017);乳糖酶缺乏组和乳糖不耐受组间比较显示,两组之间在胎龄、出生体重、年龄等所有研究因素方面均无显著性差异(P>0.017);乳糖酶不缺乏组和乳糖不耐受组间比较显示胎龄25-33周、出生体重、激素、贫血、呼吸支持、呼吸窘迫、抗生素7个方面,两组间存在显著性差异(P<0.017)。②logistic回归分析,胎龄25-33周是新生儿乳糖酶缺乏的独立危险因素(P<0.01);胎龄25-33周是新生儿乳糖不耐受的独立危险因素(P<0.01)。结论胎龄是乳糖酶缺乏及乳糖不耐受的高危因素,胎龄越小风险越高。

乳糖酶的酶学特性及其研究进展

乳糖酶在特定条件下可将乳糖水解成葡萄糖和半乳糖,对人体健康有重要作用。本文简要介绍了乳糖酶的酶学特性、生理功能以及生产应用现状,并展望了乳糖酶的发展前景。

中国儿童乳糖不耐受发生率的调查研究

随机选择北京、上海、广州和哈尔滨４大城市的３～１３岁健康儿童１１６８名，采用乳制品摄入量频率调查、乳糖耐量试验和氢呼气试验的方法，研究了中国儿童乳糖酶缺乏和乳糖不耐受的发生率。结果表明：３～５岁，７～８岁和１１～１３岁组儿童中，乳糖酶缺乏的发生率分别为３８．５％、８７．６％和８７．８％。乳糖不耐受发生率分别为１２．２％、３２．２％和２９．０％。中国４大城市儿童乳糖酶降低或消失发生的年龄在７～８岁。当儿童食用５０ｇ奶粉（含１３～１４ｇ乳糖）时，吸收不良和不耐受的发生率显著下降，但学龄儿童中仍有３９％～４１．７％为吸收不良、牛奶不耐受症状发生率１４％～１６．７％。研究未发现乳糖酶缺乏的发生和儿童的喂养史。

微生物乳糖酶研究进展

乳糖酶是一种分解乳糖产生半乳糖和葡萄糖的双糖酶,在乳品生产、医药助消化、果菜成熟软化、环境保护等方面具有重大应用价值。微生物是产乳糖酶的主要来源,具有生长繁殖快、酶量丰富、生产成本低、适合工业化生产等特点。通过查阅国内外文献,对微生物乳糖酶研究从微生物乳糖酶特点、产乳糖酶主要微生物、基因工程技术在乳糖酶生产上的应用及微生物乳糖酶的应用等领域进行了综述,为产乳糖酶微生物的开发、乳糖酶基因工程技术的应用等提供参考。

原生质体诱变选育乳糖酶高产菌株

采用紫外线诱变和60Co-γ射线协同诱变的方法,对出发菌株Uco-3的原生质体进行诱变处理,通过正突变率与诱变剂量的相互关系,确定最佳诱变剂量。采用4min的紫外线照射和剂量为500Gy的γ射线对黑曲霉Uco-3的原生质体进行诱变,获得一株产高温乳糖酶的高产突变株,突变株产乳糖酶能力显著提高,产酶活力达44.37U/mL,是出发菌株Uco-3的2.73倍。