大米淀粉水解乳酸菌的筛选

大米淀粉水解乳酸菌的筛选 Screening of Amylolytic Lactic Acid bacteria for Rice Starch Nalwoga Mariam, Liu Xiaoming, Zhang Qiuxiang, Zhang Hao, Chen Wei (School of Food Science and Technology, Jangnan University, JiangSu WuXi 214122) 5 Abstract: Twenty-nine Lactobacillus Plantarum strains potentially useful in the development of a functional ready to drink rice beverage were screened for amylolytic activity by investigating their capacities to utilize rice starch, using the known amylolytic L. plantarum A6 as a reference strain. Among these strains, 21 were capable of degrading rice starch, 10 showed extracellular amylase activity greater than 0.30 μ Units /ml. The microbial growth, titratable acidity, pH, reducing sugars and organic acids were measured respectively. Only 4 strains were able to grow substantially in the liquid 10 medium, reaching maximum cell count after 12 h fermentation (7.0-10.6 log cfu /ml), with a pH below 4.5. Lactic acid was produced in amounts ranging from 5.98 mg/ml to 8.16 mg/ml and acetic acid ranged between 3.47mg/ml to 5.90 mg/ml. Some strains produced more acetic acid than lactic acid between 12- 24 h of fermentation. L. Plantarum Z4 showed highest residual reducing sugars utilisation content. Two of the strains tested (L. plantarum Z4 and L. plantarum CS) that showed the highest 15 growth and amylase activity were recommended suitable for further studies and the development of a fermented rice beverage. Keywords: amylolytic; cereals; fermentation; lactic acid bacteria; rice; starch 20 0 Introduction The development of and demand for ready-to-drink cereal based functional foods is increasingly growing in recent research studies scientifically and technologically due to issues with household production, allergenicity, desire for vegetarian alternatives, etc. Current technological innovations include finding solutions for the stability and viability problems of multifunctional strains in new food environments that contain no milk, such as fruits, cereals, and 25 other vegetables. Therefore, researches are important to develop new media for probiotic growth and development, increasing the number of products with functionality in the market place, and offering new options for all types of consumer’s demand and desire. There is also a general agreement on the dominance and beneficial effects of lactic acid bacteria (LAB) in the fermentation processes of starchy food products in Africa and Asia[1]. Such foods are prepared 30 from cereal grains low in readily fermentable sugars and consisting mainly of starch, which would require enzymes or the use of amylolytic lactic acid bacteria (ALAB) capable of degrading starch[2][3]. Several attempts have been made to ferment cereals as individual food bases using Amylolytic lactic acid bacteria (ALAB)[1][4][5][6][7]. However, there are still few lactic acid strains 35 available that can hydrolyze starch to meet the ever-increasing demand for fermented cereal beverages in urban areas[8]. Rice (Oryzae Sativa) is a major food for over 64.24 million people in Asia and Africa, who consume it mostly in the form of fermented or unfermented preparations[9]. Fermented porridges prepared at the household level, are the most common types of food prepared from cereals[10]. A 40 wide variety of lactic acid bacteria (LAB) are found in these cereal products[11][12]. Fermentation plays an important role in most cereal food preparations, as it provides an improvement of nutritional quality, digestibility of cereal proteins[13][14], enhancement of carbohydrates accessibility[15][16], improvement of amino acid balance[13][17][18], as well as decrease of anti-nutritional factors, like tannins and phytic acid[19] and increase of vitamin content[20]. 45 Brief author introduction:Nalwoga Mariam,(1980-), F, Master, Biochemistry. Correspondance author: Chen wei,(1966-), Male, professor, Food Science and Technology. E-mail: chenwei66@jiangnan.edu.cn -1- Household fermentation technologies have been continuously upgraded to an industrial scale in order to provide value-added products that meet urban population demand for traditional products[21][13]. At the industrial level, short fermentation times are preferable in order to increase plant output as well as reduce unwanted contaminating microorganisms, which would require the 50 use of starter cultures. Being adapted to the substrate, a typical starter facilitates improved control of a fermentation process and predictability of its products[22][13], thereby avoiding variations in the quality and wholesomeness of the fermented products. As such, the development of efficient multifunctional starter cultures is a pre-requisite for the establishment of small-scale industrial production of fermented cereal foods and beverages[23][24][1][6]. Lactobacillus plantarum was 55 selected for the study because they are well documented strains for which molecular and physiological studies are available and have shown amylolytic potential in some cereals like maize, [25][5][6][7]. The aim of this study was to screen suitable probiotic millet, sorghum, and oats Lactobacillus plantarum strains with amylolytic activity for application in the development of a small-scale industrial production of a fermented rice beverage in Asia and Africa. Previous researches only screened strains for amylolytic activity without taking into consideration other 60 functional properties of the strains. 1 Material and methods 1.1 Growth conditions Twenty-nine Lactobacillus plantarum strains were obtained from the culture collection centre of Biotechnology, School of Food Science and Technology (Jiangnan University, Wuxi China). 65 All the species were originally in traditionally fermented sourdough products, camels’ intestines and milk from Inner Mongolia (China). Amylolytic Lactobacillus Plantarum A6, provided by Institut de Recherché pour le Developement (IRD) (Montpellier, France), was used as a standard strain. All the strains were preserved at -80?C in 40% glycerol throughout the course of the study. The bacterial strains were individually sub-cultured from stocks stored at -20?C in 10 ml of 70 MRS broth. MRS contained (per liter) 10 g peptone fish, 5 g yeast extract, 10 g beef extract, 20 g glucose, 2.6 g dipotassium phosphate, 2.0 g potassium dihydrogen phosphate, 2 g diammonium hydrogen citrate, 5.0 g sodium acetate, 1 ml Tween 80, 0.1 g magnesium sulfate heptahydrate, 0.05 g manganese sulphate heptahydrate. Modified MRS contained all the chemicals above, excluding beef extract, while replacing glucose with 1% rice starch purchased from Sigma 75 (Shanghai, P.R.China). 1.2 Starch Hdrolysis 1.2.1 Qualitative starch hydrolysis test for microorganisms The strains were streaked on to individual MRS agar plates and incubated at 37?C. From each 80 plate, isolated colonies were picked up and streaked in straight lines in starch agar plates with starch as the only carbon source. After incubation at 37?C for 24-48 h, individual plates were 2), 2 g potassium flooded with Gram’s iodine (Gram’s iodine solution contained 1 g iodine (I iodide (KI), 300 ml distilled water stored at room temperature) to produce a deep blue colored starch-iodine complex[26][27]. If a strain is amylolytic, it starts hydrolyzing the starch present in the plate and results in decolorization. The zone of decolorization becomes visible within a few 85 seconds of addition of I2-KI solution. The strains with amylolytic activity were used for further assays. -2- 1.2.2 Assay of α-amylase activity Extracellular α-Amylase activity of the strains was assayed by degradation of starch through 90measurement of the iodine-complexing ability in modified MRS broth under the conditions [28][29]. After the growth of L. plantarum strains in MRS-starch described by previous researchers liquid medium at 37?C for 24 h, the cells were harvested by centrifugation at 3000 X g for 15 min. The supernatant was collected and the pellets were washed twice with potassium phosphate buffer (PBS) at pH 7.2 and suspended in the same buffer. Both fractions were stored at -20?C until the amylase activity assay was conducted. The mixture containing 0.5 ml of 1% rice starch and 0.25 95 ml of 0.1 M citrate buffer (pH 5.5), was pre-warmed at 50?C for 5 min. The supernatant containing the enzymes was added to the pre-warmed solution and incubated at 50?C for 2 h. The reaction was terminated by adding 3 ml of dinitrosalicylic acid solution (DNS). After boiling for 5 min in a water bath, the absorbance of the colored solution was measured at 550 nm. One unit (U) of amylase activity was defined as the amount of enzyme that produced reducing sugar equivalent to 100 1 μ ml of glucose from starch per min at 50?C. 1.3 Measurement of pH and titratable acidity Of the 21 strains above, 10 strains with amylase activity greater than 0.30 μ Units/ml were subsequently tested for pH and titratable acidity (TA). The pH of the modified MRS broth medium for each strain described above was measured after 12, 24, 36 and 48 h using a pH meter 105 (Corning Inc., New York, USA) with a combined glass electrode and temperature probe, which was calibrated using buffers of pH 4.00 and 7.00 (Merck). For the determination of titratable acidity, 10 ml of each culture broth was titrated with 0.1 N sodium hydroxide, required to neutralize to an end point of pH 8.2. The results were expressed in % lactic acid, from which the value at time 0 had been subtracted. Standardization of sodium hydroxide was done using 0.1N 110 oxalic acid, for which 10 ml of 0.1N sodium hydroxide was taken in a conical flask and titrated against 0.1N standard oxalic acid using phenolphthalein as indicator. 1.4 Residual reducing sugar determination Reducing sugars were determined by the 3, 5-dinitrosalicylic acid (DNS) colorimetric method, with glucose as the standard[30]. About 1ml of DNS reagent was added to 1ml of sample 115 supernatant. The mixture was kept in a boiling water bath for 5 min. After cooling to room temperature (25?C) in a cold water-bath, 10 ml of distilled water was added. The absorbance was measured using a spectrophotometer (Shimadzu UV-160A Tokyo, Japan) at 575 nm wavelength, and the obtained values were interpolated with calculated values for glucose solutions of known concentration. The blanks were prepared by substituting sample solution for distilled water. 120 1.5 Organic acids determination Acetic acid and lactic acid in culture broth were analyzed according to methods described by previous researchers [31][32] using HPLC (Waters 600 Controller and Waters 996 photodiode Array Detector; Waters Associates, Inc., Milford, MA, USA). Two ml of sample were added to 6 ml of acetonitrile and 2 ml of deionized water and centrifuged at 5000 X g for 10 min. The supernatant 125 was filtered through a 0.22 μm membrane filter and stored at -20?C until HPLC analysis was conducted. An organic column packed with 9 mm of a polystyrene divinyl benzene ion exchange resin (Aminex HPX-87H; 300 mm 37.8 mm, Bio- Rad Laboratories, Richmond, CA, USA) and guard column with disposable cartridge H1 (Bio-Rad) maintained at 65?C were used for the analysis. The UV detector set at 220 nm and the mobile phase was 0.009 N sulphuric acid with a 130 flow rate of 0.7 ml/min. -3- 1.6 Growth enumeration Bacterial growth of the strains was followed by measuring viable cell enumeration. One ml of sample was serially diluted in diluents and 20 μl of the appropriate dilution were plated onto MRS 135 Agar in triplicates. The plates were incubated aerobically at 37?C for 48 h. 1.7 Statistical analysis The values are expressed as means and standard deviation for three replicates. Mean values of treatments were compared by Student’s t test. Differences were considered significant at p< 0.05 using the one-way ANOVA analysis. 140 2 Results and Discussion 2.1 Amylase activity of the various amylolytic strains Of the 29 strains tested, 21 colonies showing large clear zones by iodine staining on Modified MRS (with rice starch) agar media were picked. The strains were then tested for extracellular amylase activity in the MRS-starch liquid media after the 24 h incubation (Table 1). Though the sizes of the clear zones on the solid medium were similar, the activity of the cells grown in the 145 liquid medium differed considerably. Eleven strains showed amylolytic activity less than 0.3 μ units /ml, whereas two strains exhibited greater than 0.9 μ units /ml in their supernatants. Among the tested strains L. plantarum Z4 and L. plantarum CS exhibited high activities close to L. plantarum A6, used as a reference ALAB because of its proven efficiency in hydrolyzing starch in various food matrices[25][5][6]. 150 There was a significant difference in amylase activity between L. plantarum A6 (p<0.05) and all the other strains but no significant difference between L. plantarum Z4 and CS (P>0.05). Table 1. The extracellular-amylase activity of the 21 Lactobacillus plantarum strains studied. L. plantarum species Amylase activity in supernatants (μ/mL) f?0.01 0.10L. plantarum N1 L. plantarum N8 0.17f ?0.01 L. plantarum N11 0.10f?0.01 L. plantarum N13 0.12f?0.01 0.26de?0.02 L. plantarum J1 0.12f?0.01 L. plantarum J2 0.12f?0.01 L. plantarum J4 L. plantarum J5 0.13f?0.00 L. plantarum J7 0.12f?0.00 L. plantarum J8 0.16f?0.01 L. plantarum J9 0.30de?0.02 L. plantarum A5 0.33d?0.03 L. plantarum A6 0.98a?0.01 L. plantarum A7 0.43c?0.01 L. plantarum 12 0.32de?0.01 L. plantarum 24 0.25de?0.01 L. plantarum 45 0.44c?0.05 L. plantarum AT 0.39cd?0.01 0.89b?0.01 L. plantarum CS 0.47c?0.02 L. plantarum LB 0.93b?0.01 L. plantarum Z4 Twenty-one strains of Lactobacillus Plantarum were grown in MRS-rice starch liquid medium for 24 h and 155 assayed for extracellular amylase activity in the (supernatant) according to Section 2. The amylase activity was calculated on the basis of the original culture volume. Results were expressed by mean ?S.D. for triplicate determinations. Means in the same column with different letters in superscript are significantly different (P<0.05). 2.2 Growth of the strains in MRS starch liquid medium 160 All of the 10 strains showing amylolytic activity were subjected to this test. After 12 h -4- fermentation, only Lactobacillus plantarum Z4 and Lactobacillus plantarum CS were able to grow, reaching log 9.46?0.01 and 8.86?0.01 cfu / ml, respectively (Fig. 1). 165 Fig. 1 Growth of L.plantarum strains Z4, CS and A6 Log (CFU/ml) over a period of 12-48h. The beginning of the stationary phase occurs between 24 and 36 h after inoculation, and its onset has been attributed to the accumulation of organic acids. Other amylolytic strains reached about 8.92 log cfu /ml at 24 h while L. plantarum Z4 reached 9.54 log cfu /ml at 24 h and 10.7 log cfu /ml at 48 h. Non-amylolytic strains showed little or no growth following incubation at 18 h. 170 In a functional probiotic product, the recommended cell count is at least 107 cells per ml or gram of product at the time of consumption[22], with at least a total daily ingestion of 106 to 109 cells for any beneficial effect to occur. While all the strains generally followed a similar growth pattern, a short lag phase was markedly evident in Lactobacillus plantarum CS. The number of cfu/ml increased exponentially between 0 and 24 h and remained approximately constant after that. 175 Lactobacillus plantarum Z4 and Lactobacillus plantarum CS showed microbial counts in solid and liquid media that exceeded to a large extent the minimum value of 107 viable cells /ml recommended, indicating that the selected strains could be used in the development of a functional ready to drink fermented rice beverage. 2.3 Change of pH, titratable acidity and organic acids 180 The pH of the fermentation media (rice starch MRS) was adjusted to 6.24 before inoculation. This is because L. plantarum has been determined to grow well between pH of 4 to 9, with the optimum being 6 to 6.4[33][7]. The pH evolution during fermentation for the different strains used is shown in Fig.2. -5- 185 Fig. 2 pH and percentage titratable acidity of L. Plantarum strains Z4, CS and A6 grown in modified rice starch MRS broth between 12 and 48h. The pH gradually decreased for the first 12 h of fermentation, and then sharply dropped until 48 h, in the exponential growth period. After 48 h of fermentation, the pH ranged between 4.48 for 190 strain L. Plantarum Z4 and 4.90 for L. Plantarum CS, except for L. Plantarum A6, where the pH after 12 h of fermentation was 4.54 and 3.75 after 48 h. Other strains tested ranged between 5.9 at 12 h to 5.6 at 48 h (data not shown). The pH values observed in this study are comparable to other works on rice fermentations[21][26][27]. In general, TA increased and the pH decreased as the fermentation time increased for rice starch medium inoculated with all the strains. L. plantarum 195 Z4 produced most acid, with TA increasing from an initial 0.025% to 0.295% after 48h. All the TA values were below the maximum limit of 0.4% acidity as lactic acid according to the International Standard, ISO 750:1998. The drop in pH is due to the production of organic acids. Theoretically, the fermentation of 2 mol of glucose by L. plantarum produces 3 mol of acetate and 2 mol of lactate via the 200 fructose-6-phosphate shunt[34]. The proportions of each metabolite depend on the carbon source, the strain used or the fermentation conditions. To be organoleptically acceptable, the pH of a fermented beverage must be between 4 and [33] 4.5 in addition growth of pathogenic microorganisms would be prevented. The general slow 205 rate to reach pH 4.5 is attributed to the growth medium, which would be lacking in some nutrients to support faster growth of the strains requiring shorter fermentation times. The presence of lactic and acetic acids in fermented foods is advantageous due to their antimicrobial properties, [35]. However, accumulation of large quantities of preventing spoilage by other microorganisms these acids may lead to inhibition of desirable microbial growth required to impose an antimicrobial effect in the food. In addition, large quantities of acetic acid are not desirable 210 because of their negative organoleptic properties. Small amounts of these acids measured (lactic acid and acetic acid) were present at the beginning of the fermentation (Fig. 3 and Fig. 4). -6- Fig. 3 Lactic acid (mg/ml) produced by L. plantarum strains CS, Z4 and A6 between 12 and 48h. 215 Fig. 4 Acetic acid (mg/ml) produced by L. plantarum strains CS, Z4 and A6 between12 and 48h. Initial concentrations of lactic acid and acetic acid were in the range of 0.094–0.102 mg/ml, and 0.032–0.056 mg/ml, respectively. The increase in the concentration of acids started to be 220 noticeable after 12 h of fermentation, and increased linearly till the end of the fermentation. Both lactic and acetic acid are produced in large amounts by L. plantarum CS and Z4, reaching concentrations of 7.98 and 4.25 mg/ml as well as 8.16 mg/ml and 3.75 mg/ml, respectively. Interestingly, the production of lactic acid slowed down after 24 h of fermentation, whereas acetic acid production continued for strain A6 and CS. This is in agreement with [36][5] about the 225 ability of L. plantarum species to convert some lactic acid into acetic acid. Given that organoleptically the presence of lactic acid is more favorable than the presence of acetic acid, L. plantarum Z4 (acetate: lactate 0.77) seems to be more appropriate for development of a lactic-fermented rice-based beverage. 2.4 Residual reducing sugars 230 Fermentation promoted a decrease in reducing sugars in almost all the samples as a result of their consumption as an energy source. The exception is for the sample fermented with L. plantarum Z4 (Fig. 5). In this sample, the increase in soluble sugars could be related to hydrolysis -7- by bacterial amylases. This assumption is confirmed by the increase in color intensity observed in 235 the sample fermented with L. plantarum Z4. Sugar solubilization promoted by this species seemed to be superior to its consumption needs. The two different trends in soluble sugars were found in [18][21][5]. other studies on rice fermentation Fig. 5 Residual reducing sugar concentration (mg/ml) of L. plantarum strains CS, Z4 and A6 between 12 and 48h. 240 In this work, only limited physiological studies were carried out since strains were selected for their amylolytic activity. All the tested strains of Lactobacillus plantarum were able to ferment rice starch. Fermentations led to organic acids production and consequent increase in titratable acidity, decrease in pH, and sugar metabolism. The results indicate that the two representative strains chosen in this study, L. plantarum strains Z4 and CS grow well in the media of interest. 245 Starch was fermented according to a now well-established pattern for different ALAB by [37][25] and characterized by a fast starch hydrolysis, a transient reducing sugar accumulation and a growth linked amylase production. However, L. plantarum strain Z4 yields and specific rates of amylase production were markedly higher than those of L. plantarum CS. These results indicate that strain Z4 was a more efficient amylase producer than strain CS. Efficient amylolysis was 250 chosen as the main criterion of strain selection, because in a starchy food matrix, it is expected to increase the availability of energy sources for other associated non-amylolytic lactic acid bacteria to contribute to a rapid pH decrease, and to impart favorable organoleptic and rheological properties. 255 3 Conclusion In conclusion, this work aimed to study the growth and amylolytic activity of different L. plantarum species in a modified rice starch MRS medium with a starch composition mimicking the one obtained in commercial rice flour. The work provides an approach to the possibility to use the chosen strains of lactic acid bacteria on an industrial scale production of rice-fermented foods with improved functional and nutritional qualities. More studies are, underway to prove in vitro 260 and in vivo the probiotic characteristics of the chosen strains, and to determine if the fermentation media exerts any protective effect to the bacteria in their passage through their gastrointestinal -8- tract prior to the development of a ready to drink fermented cereal beverage. Acknowledgements 265 The authors would like to acknowledge the support provided in terms of standard strain, L. plantarum A6, by Dr. Jean Pierri Guyot from Institut de Recherché pour le Developpement (IRD) and paper editing by Dr. Stephanie Clark from Iowa State University. This work was supported by the National Science Fund for Distinguished Young Scholars (No. 31125021), the National High Technology Research and Development Program of China (863 Program No. 2011AA100901), the National Natural Science Foundation of China (No. 31200691), and the 111 project B07029 270 References [1] Sanni A, Morlon-Guyot J, Guyot J P. 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[37] Guyot J P, Morlon Guyot J.Effect of different cultivation conditions on lactobacillus manihotivorans OND 355 32T, an amylolytic lactobacillus isolated from sour starch cassava fermentation[J]Food Microbiology, 2001,67:217-225. 大米淀粉水解乳酸菌的筛选 玛丽，刘小鸣，张秋香，张灏，陈卫 360 (江南大学食品学院，江苏 无锡 214122) 摘要:本研究对 29 株能够用于大米发酵饮料的植物乳杆菌进行筛选，利用 L. plantarum A6 淀粉水解菌作为参照菌株，通过比较 29 株乳酸菌水解大米淀粉的能力，筛选出具有较高淀 粉水解活性的乳酸菌。研究 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 明:在 29 株菌株中，有 21 株能够降解大米淀粉，其中有 10 株胞外淀粉酶活力高于 0.3μ U /ml。对微生物的生长，滴定酸度，pH 值，还原糖和有机酸 365 分别进行研究，结果表明:有 4 株菌株能够在液体培养基中大量生长，在 pH 低于 4.5 的条 件下发酵 12 小时，能够达到最大菌落数(7.0-10.6 log cfu /ml)，乳酸产量为 5.98 mg/ml-8.16 mg/ml，醋酸产量为 3.47mg/ml - 5.90 mg/ml，有些菌株在 12-24 小时的发酵培养中产生的醋 酸大于乳酸。在这 29 株菌株中，L. Plantarum Z4 菌具有最高的还原糖利用力。在所研究的 29 株菌株中，L. plantarum Z4 和 L. plantarum CS 具有最强生长能力和最高的淀粉酶活性， 370 适于进行进一步研究，以满足在功能性发酵米酒中应用的要求。 关键词:淀粉水解;谷物;发酵;乳酸菌;大米;淀粉 中图分类号:Q936》 - 10 -