Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains

In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.     

Short communication: Effect of kefir grains on proteolysis of major milk proteins

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse phase-HPLC analysis. The reduction of κ-, α-, and β-caseins (CN), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) contents during 48 and 90 h of incubation of pasteurized milk (100 mL) and respective cheese whey with kefir grains (6 and 12 g) at 20°C was monitored. Significant proteolysis of α-LA and κ-, α-, and β-caseins was observed. The effect of kefir amount (6 and 12 g/100 mL) was significant for α-LA and α- and β-CN. α-Lactalbumin and β-CN were more easily hydrolyzed than α-CN. No significant reduction was observed with respect to β-LG concentration for 6 and 12 g of kefir in 100 mL of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of α-LA and β-LG: α-LA was hydrolyzed between 60 and 90% after 12 h (for 6 and 12 g of kefir) and no significant β-LG proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of α-LA and κ-, α-, and β-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. β-Lactoglobulin is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase β-LG digestibility in whey-based or whey-containing foods.     

微囊化纯培养发酵剂发酵奶与藏灵菇奶的比较研究

利用从藏灵菇中分离出的优势菌,进行混合发酵,比较了这种纯培养菌种发酵的酸奶与藏灵菇发酵奶在感官、主要风味成分以及优势菌株数量的差异。HPLC检测结果表明,这2种发酵奶的乳酸产量分别为7.62 g/L和7.85 g/L,无显著差异;GC检测结果表明,微囊化纯培养发酵剂发酵奶中丁二酮的含量明显高于传统藏灵菇奶;GC-MS检测结果表明,2种发酵奶中挥发性风味成分有90%相同;感官评定表明微囊化纯培养发酵剂发酵奶的可接受程度显著高于藏灵菇奶。表明,微囊化纯培养发酵剂发酵奶既保持了藏灵菇奶原有的特征,又在风味上得到了进一步的提高,表明微囊化纯培养发酵剂可以替代藏灵菇进行这类发酵奶的规模化生产。     

Probiotic potential and biochemical and technological properties of Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains

Lactococcus lactis ssp. lactis is one of the most important starter bacteria used in dairy technology and it is of great economic importance because of its use in the production of dairy products, including cheese, butter, cream, and fermented milks. Numerous studies have evaluated the biochemical and probiotic properties of lactococci; however, limited studies on the probiotic characteristics of lactococci were conducted using strains originating from raw milk and dairy products. Characterizing the probiotic properties of strains isolated from raw milk and fermented milk products is important in terms of selecting starter culture strains for the production of functional dairy products. In this study, biochemical properties (including antibiotic sensitivity, lipolytic activity, amino acid decarboxylation, antioxidant activity) and probiotic properties (including antimicrobial activity, growth in the presence of bile salts, bile salts deconjugation, and hydrophobicity) of 14 Lactococcus lactis strains isolated from raw milk and kefir grains were investigated. Strains originating from kefir grains had better characteristics in terms of antimicrobial activity and bile salt deconjugation, whereas strains from raw milk had better hydrophobicity and antioxidant activity characteristics. None of the strains were able to grow in the presence of bile salt and did not show amino acid decarboxylation or lipolytic activities. Biochemical and probiotic properties of L. lactis strains varied depending on the strain and some of these strains could be used as functional cultures depending on their properties. However, these strains did not possess all of the properties required to meet the definition of a probiotic.     

Characterisation of the exopolysaccharide kefiran produced by lactic acid bacteria entrapped within natural kefir grains

The main goal of this work was to characterise and quantify the exopolysaccharide kefiran and to discover an effective procedure for its isolation from kefir grains, originating from the Caucasian Mountains. Capillary electrophoresis was used for the characterisation and quantification of the d ‐glucose and d ‐galactose in our samples at a mass ratio of 1:0.7. The effect of fermentation time on growth of kefir grains and the content of kefiran within the grains were determined. The pH profiles were monitored dynamically. In addition, the influence of fermentation temperature on kefir grains mass concentration (γ_KG) and the content of kefiran within the grains (w_KEF/KG) were studied. The highest values for both were obtained at 30 °C.     

水开菲尔粒发酵香蕉皮过程中成分变化研究

采用水开菲尔粒分别发酵蒸煮和未蒸煮香蕉皮,按红糖、香蕉皮、水、水开菲尔粒1:3∶10∶0.5的质量比于28 ℃发酵。 结果表明, 在发酵初期48 h内,发酵液的pH值及糖度均迅速下降,此后显著变缓。 240 h后测得未蒸煮和蒸煮（100 ℃蒸煮15 min）香蕉皮的利用 率分别为32.4%和43.8%;发酵液的pH值分别为3.53和3.51;滴定酸度分别为50.91 mmol/100 mL和54.89 mmol/100 mL;乙醇含量分 别为7.90 mg/mL和2.80 mg/mL;抗氧化能力（ORAC）值分别为5.03 mmol Trolox/L和3.58 mmol Trolox/L。 香蕉皮原料的挥发性成分主 要是丁酸酯类,发酵后这些成分全部消失,并产生大量新的挥发性成分,包括乙酸异戊酯、辛酸乙酯、芳樟醇等芳香成分。所得发酵液 色泽类似红茶水,风味类似果醋,具浓郁果香。 蒸煮香蕉皮适合用水开菲尔粒酿制果醋。     

开菲尔核桃乳饮料的研制

以开菲尔粒为发酵剂发酵核桃乳。选择影响开菲尔品质的四个因素:发酵温度、发酵时间、接种量和蔗糖用量设计实验。通过正交实验L9(34),来确定发酵开菲尔核桃乳饮料的最佳工艺条件。结果表明,最佳发酵条件为发酵温度30℃,发酵时间12h,接种量5%,蔗糖用量8%。用这种方法制备的开菲尔核桃乳饮料口感细腻,风味独特。     

开菲尔酸豆乳冰淇淋的研制

以豆乳代替部分牛乳并用开菲尔进行发酵,研制出开菲尔酸豆乳冰淇淋。选择煮浆时间、豆粉奶粉比例、白砂糖添加量、复合乳化稳定剂添加量、老化时间和凝冻档位作为单因素进行梯度实验,对发酵时间、接种量和发酵温度进行三因素三水平L9（33）正交实验,确定开菲尔酸豆乳冰淇淋的最佳工艺为:取浸泡好的大豆,与水按照1∶11的比例进行磨浆,过滤,沸腾后煮浆5 min,制得豆乳;以豆浆重量为基准,加入8%豆粉、5%奶粉、16%白砂糖和0.65%复合乳化稳定剂,倒入制浆机中搅拌并杀菌,待均质后的料液冷却到38℃加入料液总质量0.2%的开菲尔发酵剂,38℃发酵8 h,得到发酵乳;将发酵乳置于4℃老化缸中老化4 h,然后将料液倒入冰淇淋机器设置成三档（凝冻温度-7℃）,制得开菲尔酸豆乳冰淇淋。      

雪莲菌发酵豇豆工艺优化及其挥发性风味成分分析

利用雪莲菌对豇豆进行发酵,以感官评分和总酸含量为评价指标,采用单因素和正交试验对发酵工艺进行优化;并采用高效液相色谱（HPLC）法和气相色谱—质谱联用（GC-MS）分析发酵前后风味物质的变化。结果表明,最佳发酵工艺条件为盐添加量4%,雪莲菌接种量1.5%,发酵温度25 ℃,发酵时间4 d ,此条件下感官评分为87分,总酸含量为3.40 g/kg。雪莲菌发酵后共检出72种挥发性风味物质,与原料相比,新产生了56种挥发性风味物质。除醛类物质外,其他挥发性风味物质相对含量均增加,且醇类和酯类含量较高。与自然发酵相比,雪莲菌发酵后的挥发性物质种类和含量也较高。     

Investigation on culturable microflora in tibetan kefir grains from different areas of china

Four samples of Tibetan kefir grains (TK-ZJUJ 01-04) from Tibet and surrounding areas were investigated via phenotypic and genotypic methods to compare and analyze the diversity of culturable microflora among different origins. As a result, 4 genera of microorganisms from TK-ZJUJ01: Bacillus subtilis (2.9 × 10(7) cfu/mL), Lactococcus lactis (8.2 × 10(7) cfu/mL), Kluyveromyces marxianus (3.0 × 10(6) cfu/mL), Saccharomyces cerevisiae (9.0 × 10(6) cfu/mL); 4 genera from TK-ZJUJ02: Lactobacillus kefiri (1.0 × 10(8) cfu/mL), Pichia kudriavzevii (5.0 × 10(6) cfu/mL), K. marxianus (1.9 × 10(7) cfu/mL), Kazachstania unispora (6.2 × 10(7) cfu/mL); 6 genera from TK-ZJUJ03: Leuconostoc lactis (4.6 × 10(7) cfu/mL), L. lactis (3.0 × 10(7) cfu/mL), Lactobacillus plantarum (3.0 × 10(7) cfu/mL), K. unispora (3.0 × 10(6) cfu/mL), K. marxianus (2.0 × 10(6) cfu/mL), (1.7 × 10(7) cfu/mL); and 4 genera from TK-ZJUJ04: L. plantarum (1.8 × 10(7) cfu/mL), Acetobacter fabarum (5.0 × 10(6) cfu/mL), K. unispora (6.2 × 10(7) cfu/mL), Pichia guilliermondii (6.2 × 10(7) cfu/mL) were identified. Yeasts like P. kudriavzevii and P. guilliermondii isolated in this study were the first time reported in Tibetan kefir grains. For TK-ZJUJ 01-03, lactic acid bacteria were the major microorganisms, which accounted for more than 50% of all the microbial population, while for TK-ZJUJ04, the largest microbial group was yeasts which accounted for more than 50%. In a word, study of diversity and composition of microflora provided us theoretical foundation for further investigation and application of Tibetan kefir grains. Practical Application: This is the basic research in order to develop and industrialize a new kind of yogurt starter which is naturally formed microbiota with both lactic acid bacteria and yeasts in it.     

Sheep milk kefir sweetened with different sugars: Sensory acceptance and consumer emotion profiling

The objective of this study was to determine the sensory acceptance and emotional profile of sheep milk kefir sweetened with different sugars (demerara sugar, brown sugar, fructose, coconut sugar, and honey, 100 g/L). Consumers (n = 100) assessed sensory acceptance (appearance, aroma, taste, texture, and overall impression), and expressed their emotions (satisfied, active, loving, calm, comfortable, energetic, happy, healthy, refreshing, disgusted, worried, and upset). The emotions “satisfied,” “active,” “comfortable,” “energetic,” “healthy,” and “refreshing” were found between moderate and very high levels, indicating that they are important emotions for the characterization and sensory acceptance of kefir samples. The use of different sugars had no influence on the intensity of the emotions “calm,” “comfortable,” “happy,” or “disgusted,” and resulted in a greater sense of satisfaction. The use of demerara sugar or fructose did not alter the acceptance of the products or the intensity of emotions. The use of brown sugar decreased acceptance (taste, texture, and overall impression) and the intensity of the emotions “active,” “loving,” “energetic,” “healthy,” and “refreshing.” The use of coconut sugar decreased acceptance (appearance, aroma, and taste) and the intensity of the emotions “refreshing” and “upset.” The use of honey improved acceptance in appearance and aroma but reduced the intensity of the emotions “active,” “loving,” “energetic,” and “healthy.” Based on sensory data, it is recommended to use demerara sugar or fructose as a substitute for sucrose. In conclusion, the study of emotions can be used as an additional tool for obtaining data related to the sensory acceptance of products.     

Comparative analyses of microbial community diversities of Tibetan kefir grains from three geographic regions

Tibetan kefir grains are the traditional starter for fermented milk products in Tibet, China. To gain a deeper understanding of their bacterial and fungal community diversities, we compared kefir grains from three different regions, Nyingchi, Lhasa and Yangbajing, using high‐throughput sequencing. The results of sequence analysis grouped the reads into four bacterial and four fungal phyla, comprising 34 bacterial and 139 fungal genera, and 44 bacterial and 195 fungal species. Firmicutes and Proteobacteria were two dominant bacterial phyla, and Lactobacillus and Acetobacter were the dominant bacterial genera. Basidiomycota and Ascomycota were the dominant fungal phyla. There was significant difference in the Basidiomycota and Ascomycota among three regions (P < 0.05). A significant difference in genera was observed in the percentage of Saccharomyces, Trichosporon, Aspergillus and Candida between the samples from Nyingchi, Lhasa and Yangbajing (P < 0.05). Based on principal component analysis (PCA), the Tibetan kefir grain samples from Nyingchi could be distinctly differentiated from the other two regions in term of fungal community, but no obvious difference existed in the bacterial population of samples from the three regions.     

Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis

The microflora of Tibetan kefir grains was investigated by culture- independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rRNA for bacteria and 26S rRNA for yeasts, followed by sequencing of the most intense bands, showed that the dominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua. The bacterial communities between three kinds of Tibetan kefir grains showed 78–84% similarity, and yeasts 80–92%. The microflora is held together in the matrix of fibrillar material composed largely of a water-insoluble polysaccharide.     

功能性开菲尔牛奶酒最佳发酵条件的研究

本文利用开菲尔粒中分离的酵母菌和乳球菌采用两步发酵法研制开菲尔牛奶酒。针对影响牛奶酒制品最终品质的三个因素,采用三因素三水平L9(34)正交试验确定了功能性开菲尔牛奶酒的最佳发酵条件。成品开菲尔牛奶酒口感细腻、酸度适中,风味柔和,有浓郁的醇香和酯香味。     

Assessment of beverages made from milk,soya milk and whey using Iranian kefir starter culture

Kefir starter culture was used for the production of beverages, and some chemical and microbiological parameter changes were determined during 24 h of fermentation and then after 2, 7, 14, 21 and 28 days of storage at 4 °C. Three different substrates (milk, whey and soya milk) were used as fermentation media for the kefir starter culture. After the fermentation, the carbohydrate content and pH decreased, but the dry matter and fat content of the beverages were not significantly different from their substrates. During storage, lactic acid bacteria in the beverages decreased, while yeasts increased and carbohydrate, ethanol and pH changed significantly in the three beverages.     

开菲尔直投式发酵剂在益生菌干酪生产中的应用研究

该研究主要将开菲尔粒制备成直投式发酵剂应用于干酪的生产。 以FD-DVS R-704商业干酪发酵剂制作的干酪为对照,对其 理化指标、挥发性香气成分、质构特性和感官指标进行测定与评价。 结果表明,与对照干酪相比,开菲尔干酪的水分含量、pH4.6-可溶 性氮（SN）及12%三氯乙酸（TCA）-SN的含量极显著增高（P＜0.01）,pH值极显著低（P＜0.01）,而粗脂肪比例和总游离氨基酸（TFAA） 含量无显著差异（P＞0.05）,不饱和脂肪酸（UFA）含量显著增高（P＜0.05）;醇类和酯类物质的种类增多;硬度和咀嚼性极显著降低（P＜0.01）,胶黏性极显著增大（P＜0.01）,内聚性和弹性无明显差异（P＞0.05）;开菲尔粒益生菌干酪呈乳白色,表面有光泽,不仅具 有干酪特有的滋味和气味,且具有开菲尔发酵乳的特有风味,带有适宜的酒香味。     

Short communication: Physicochemical features and microbial community of milk kefir using a potential probiotic Saccharomyces cerevisiae KU200284

The aim of this study was to analyze the β-glucan contents, physicochemical features, and microbial communities in milk kefir prepared using Saccharomyces cerevisiae KU200284 isolated from cucumber jangajji, a fermented vegetable commonly eaten in Korean. Three types of milk kefir were manufactured, with (1) activated kefir grain, (2) activated kefir grain with commercial S. cerevisiae BOF, and (3) activated kefir grain with S. cerevisiae KU200284. β-Glucan contents of milk kefir using kefir grain and kefir grain with S. cerevisiae strains BOF and KU200284 were 8.29, 8.59, and 8.57%, respectively. The pH, titratable acidity, viscosity, Brix level, and alcohol contents of milk kefir using kefir grain with S. cerevisiae strains were acceptable compared with milk kefir using only kefir grain. In milk kefir produced using kefir grains and S. cerevisiae strains, 16S rRNA reads showed representative strains of Lactobacillus kefiranofaciens (>72% relative abundance) and Acetobacter fabarum (>16% relative abundance). In particular, milk kefir using kefir grain with S. cerevisiae KU200284 had the highest relative abundance of L. kefiranofaciens. In addition, the internal transcribed sequence (ITS) rRNA reads in tested milk kefir showed representative strains of Kluyveromyces marxianus (>52% relative abundance) and Saccharomyces cerevisiae (>16% relative abundance). In contrast, milk kefir using S. cerevisiae strains had higher relative abundance of S. cerevisiae (>37%). The β-glucan production, physicochemical properties, and microbial community profiling indicate that S. cerevisiae KU200284 could be used in functional dairy products as a starter culture.