【作者】 张清丽；

【导师】 赵谋明；

【作者基本信息】 华南理工大学 ， 食品科学， 2011， 博士

【摘要】 由于乳酸菌缺乏某些生物合成途径,不能合成其生长所必需的一些维生素和氨基酸等物质,对营养要求苛刻。另一方面由于乳酸菌对蛋白的水解能力有限,牛乳中游离氨基酸及寡肽含量很低,导致乳酸菌在牛乳中生长缓慢,造成酸乳发酵时间较长。嗜热链球菌(球菌)和德氏乳杆菌保加利亚亚种(杆菌)是酸乳最常用的发酵剂菌株。本文以酪蛋白为原料,通过五种的蛋白酶(碱性蛋白酶、风味蛋白酶、中性蛋白酶、木瓜蛋白酶和胰蛋白酶)进行酶解并对其酶解特性进行了研究,利用促进乳酸菌(球菌和杆菌1:1)生长活性实验评价了五种酪蛋白水解物的生物活性。从乳酸菌生长代谢机制出发,考察了不同酪蛋白酶解物对乳酸菌生长状态、产乳酸、生物合成胞外多糖(EPS)及乳酸菌发酵酸乳的影响。进一步研究了酪蛋白酶解物在乳酸菌发酵过程中对涉及乳酸菌乳酸生物合成途径,即糖酵解途径和EPS生物合成途径中关键酶活性的影响。最后,利用膜分离、大孔吸附树脂色谱、凝胶渗透色谱和高效液相色谱(HPLC)一系列分离技术从活性最强的酪蛋白酶解物中分离纯化得到了具有促乳酸菌生长活性的酪蛋白肽,并利用液相色谱质谱联用(HPLC-MS/MS)技术结合氨基酸分析对纯化后的肽进行了一级序列结构鉴定。分别采用碱性蛋白酶、风味蛋白酶、中性蛋白酶、木瓜蛋白酶和胰蛋白酶对酪蛋白进行酶解,以促进乳酸菌生长活性为指标,筛选出各种蛋白酶的最佳水解条件,得到促生长活性最好的酪蛋白水解物。进一步将酪蛋白酶解物进行膜分离,得到分子量段不同的酪蛋白水解物,并评价了不同分子量段的酪蛋白水解物促乳酸菌的增殖活性。研究了不同蛋白酶、水解度、分子量和氨基酸组成对水解物的促乳酸菌增殖作用的影响。结果表明,酪蛋白水解物的水解度与促生长活性没有明显的相关性;酪蛋白分别用碱性蛋白酶、风味蛋白酶、中性蛋白酶和胰蛋白酶进行水解12 h(CHA、CHF、CHN和CHT)以及用木瓜蛋白酶水解8 h (CHP)得到促生长活性较强的水解物;分子量(Mw)小于3 kDa段的各种酪蛋白活性肽(CHA-Ⅳ、CHF-Ⅳ、CHN-Ⅳ、CHP-Ⅳ和CHT-Ⅳ)具有比其它分子量段更好的促生长活性;酪蛋白水解物中富含的亲水性氨基酸(组氨酸、赖氨酸、谷氨酸和丝氨酸)对乳酸菌的生长具有更好促生长作用,而含硫氨基酸(半胱氨酸和甲硫氨酸)对水解物的促生长活性没有明显的影响。研究了五种Mw < 3 kDa段酪蛋白水解物(CHA-Ⅳ、CHF-Ⅳ、CHN-Ⅳ、CHP-Ⅳ和CHT-Ⅳ)对乳酸菌在72 h发酵过程中乳酸菌活菌数、乳酸生成、葡萄糖消耗以及糖酵解关键酶(葡萄糖激酶、磷酸果糖激酶、丙酮酸激酶和乳酸脱氢酶)活性的影响。结果表明,五种酪蛋白活性肽段(Mw < 3 kDa)的添加均能够提高球菌的活菌数,尤其是在对数生长期的影响更显著,而对此阶段杆菌活菌数没有太大的影响;酪蛋白活性肽能够减缓乳酸菌活菌数在生长后期的下降趋势;对乳酸菌发酵液菌体量都有不同程度的提高作用,其中CHP-Ⅳ的影响最显著;与此同时,由于各种酪蛋白肽段的添加发酵液pH下降趋势均有不同程度的加剧,加速了发酵过程葡萄糖的消耗速度,其中以CHP-Ⅳ的影响最明显;酪蛋白活性肽段的添加明显提高了丙酮酸激酶和磷酸果糖激酶这两种酶活性,但是对葡萄糖激酶和乳酸脱氢酶的酶活性影响不大,并且,丙酮酸激酶和磷酸果糖激酶的酶活性与乳酸菌产乳酸量有很好的相关性。考察了CHA-Ⅳ、CHF-Ⅳ、CHN-Ⅳ、CHP-Ⅳ和CHT-Ⅳ对乳酸菌72 h发酵过程中胞外多糖(EPS)产量、单糖组分和EPS合成途径中关键酶(α-磷酸葡糖糖变位酶、β-磷酸葡糖糖变位酶、TDP-葡糖糖焦磷酸化酶、UDP-葡糖糖焦磷酸化酶和UDP-半乳糖-4-异构酶)活性的影响。结果表明,五种酪蛋白活性肽段(Mw < 3 kDa)的添加都能提高乳酸菌EPS产量,但对EPS产量的影响不同;乳酸菌EPS主要是由葡萄糖、半乳糖和鼠李糖组成,其单糖组成比例随着发酵时间的变化而有所不同,各种酪蛋白活性肽段(Mw < 3 kDa)的添加对EPS单糖组成比例没有影响;α-磷酸葡萄糖变位酶、TDP-葡萄糖焦磷酸化酶和UDP-葡萄糖焦磷酸化酶影响乳酸菌EPS发酵过程中的产量。UDP-半乳糖-4-异构酶和β-磷酸酪蛋白活性肽段(Mw < 3 kDa)变位酶对EPS产量没有影响。Mw < 3 kDa段酪蛋白活性肽的添加能够提高α-磷酸葡糖糖变位酶、TDP-葡糖糖焦磷酸化酶、UDP-葡糖糖焦磷酸化酶和UDP-半乳糖-4-异构酶这四种酶活性,而对β-磷酸葡糖糖变位酶活性没有影响。研究了Mw < 3 kDa段木瓜蛋白酶水解酪蛋白活性肽(CHP-Ⅳ)对酸乳发酵过程的影响(发酵过程中pH值,凝乳时间,产酸量和产酸速度以及发酵时间等发酵参数)以及对发酵乳储藏期品质的影响,包括对发酵乳质构特性、稳定性、后酸化和乳酸菌活性变化的影响。结果表明,在一定添加量范围内(1.5-3.0%等量蛋白替代, w/w),CHP-Ⅳ对酸乳促发酵能力随着添加量的增加而增强。在3.0%(w/w)的等量蛋白替代下,酸乳发酵时间缩短了21.9%;超过了3.0%(w/w)的添加量,即在6.0%(w/w)的添加量下,该水解物的促发酵效果不明显;在3.0%(w/w)的添加量下能够明显延缓酸乳在储藏期的后酸化;能够明显提高酸乳储藏期间球菌的活性,但是对杆菌的活性有轻微的抑制作用;该水解物不仅能够明显提高酸乳储藏期间的活菌总数而且还改变了球/杆菌数比例;对储藏期酸乳的硬度没有明显的改变,但是可以提高酸乳的粘度,明显提高酸乳的感官质量。通过大孔吸附树脂色谱、凝胶渗透色谱和反相高效液相色谱等系列分离技术,分别根据酪蛋白肽的亲疏水性、分子量大小和极性大小等性质的不同对Mw < 3 kDa段的酪蛋白木瓜蛋白酶水解物(CHP-Ⅳ)进行逐级分离纯化,同时评价了各级分离产物的促乳酸菌生长活性,以HPLC-MS/MS结合氨基酸分析对纯化得到的功能肽的氨基酸序列结构进行鉴定,首次分离得到三个具有显著促乳酸菌生长活性的酪蛋白肽CHP-Ⅳ-F1-G1-H2 (Asn-Pro-Ser-Lys-Glu-Asn-Leu)、CHP-Ⅳ-F2-P2-Hb (Asp-Ile-Pro-Asn- Pro-Ile)和CHP-Ⅳ-F2-P2-Hc (Pro-Ile-Val-Leu-Asn-Pro)。更多还原

【Abstract】 A characteristic of lactic acid bacteria (LAB) is their fastidious requirements for growth and biological activities, particularly amino acids. The pool of free amino acids and peptides in milk is not enough to guarantee optimum bacterial growth in this substrate. Lactobacillus delbrueckii subsp. bulgaricus (LB) and Streptococcus thermophilus (ST) are the most comMon yoghurt starter. On the basis of these findings, we reported a new promising nitrogen source, casein hydrolysates. Casein protein was respectively hydrolyzed with different proteolytic enzymes (alcalase, flavorzyme, neutrase, papain and trypsin) and the effect of ultrafiltered fractions with different molecular mass from five casein hydrolysates (CH) on the growth performance and lactic acid production of LAB (St and Lb 1:1) in MRS broth medium was determined. Besides, the influence of five peptides fractions (< 3 kDa) of CH on the production of lactic acid and exopolysaccharides (EPS), glucose consumption, EPS monomeric composition, the enzymatic activities of LAB and yoghurt fermentation was evaluated. Finally, the peptides with growth-promoting activity for LAB originated by CH was isolate and purified using ultrafiltration, macroporous adsorption resin chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC), sequentially. And, the purified peptides were identified by HPLC-ESI-MS/MS.The growth performance of Lb and St was determined in the presence of casein hydrolysates produced by the action of five proteolytic enzymes (alcalase, flavorzyme, neutrase, papain and trypsin) with various degree of hydrolysis (DH). The CH respectively treated with alcalase, flavorzyme, neutrase, and Trypsin for 12 h and the CHs (CHA, CHF, CHN and CHT) with high growth-promoting activity were obtained. The casein was hydrolyzed by papain for 8 h (CHP) to obtain the hydrolysates with high growth-promoting activity. In addition, these five kinds of CHs were fractionated by ultrafiltration and the influence of the amino acid composition of the peptides fractions on the growth and lactic acid yield of LAB was studied. The results showed that the ultrafiltered fractions with molecular mass less than 3 kDa (CHA-IV, CHF-IV, CHN-IV, CHP-IV and CHT-IV) were the determinant stimulator in crude hydrolysates, respectively. Furthermore, the hydrophilic amino acid residua including His, Lys, Glu and Ser were beneficial for bacterial growth.The influence of CHA-IV, CHF-IV, CHN-IV, CHP-IV and CHT-IV on cell viability, lactic acid production, glucose consumption and the activities of enzymes (glucokinase, phosphofrutokinase, pyruvate kinase and lactate dehydrogenase) involved in the glycolysis pathway in the mixed cultures of St and Lb. The viability of St was respectively improved by all the peptides fractions, especially during the exponential growth phase, but there was no enhancement for Lb was observed. The addition of the peptides fractions could slow down the decrease in bacterial viability during the decline phase. Besides, the cell density of LAB could be improved by all the fractions, especially CHP-IV. The consumption of glucose by LAB was inordinately exacerbated by the different addition. The activities of phosphofrutokinase and pyruvatekinase were improved by the addition of peptides fractions, but the activities of glucokinase and lactate dehydrogenase were not influenced. In addition, there was a good correlation between lactic acid production and the activities of phosphofrutokinase and pyruvatekinase.The influence of different CH (< 3 kDa) on EPS production and the activity of the enzymes involved in the synthesis of EPS by LAB was evaluated. The highest EPS production (138.2 mg/l) was obtained on CHP-IV at 24 h when the EPS produced in all fermentations was composed of galacose, glucose and rhamnose in a ratio of 1.0: 2.4: 1.5. The sugar composition just changed according to the fermentation times. The activity ofα-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase was associated with EPS production. Moreover, the activity ofβ-phosphoglucomutase and dTDP-glucose pyrophosphorylase implicated in rhamnose synthesis was very low at exponential growth phase and could not be detected at other given period.The effect of CHP-IV on the fermentation kinetics, microbiological survival and physicochemical properties in yoghurt was determined. The value of TpH4.5 (time necessary to reach pH 4.5) of yoghurt fortified with CHP-IV reduced from 5.21 h to 4.29 h and the total counts of LAB present in the final products was increased by the addition at nitrogen replaced ratio of 3.0% (w/w). Moreover, the yoghurt exhibited lower post-acidification power, higher viscosity and sensory evaluation scores than did the control product during storage period.A series of separation methods including ultrafiltration, macroporous adsorption resin chromatography, gel filtration chromatography and HPLC were applied to isolate and purify the peptide(s), which were mainly responsible for the activity. Finally, three novel growth-stimulating peptides, CHP-IV-F1-G1-H2, CHP-IV-F2-P2-Hc and CHP-IV -F2-P2-Hb corresponding to amino acid residues 29-35 and 103-108 of bovineαS2–casein and 181-186 of bovineαS1–casein, respectively, were obtained.更多还原