Development technology of starter cultures using lactic acid bacteria isolated from fermented Camel milk with cholesterol lowering ability

9 The aim of the study is to develop a technology of starter cultures for fermented milk using new strains of lactic acid bacteria isolated from Mongolian traditional fermented camel milk. “Khoormog” samples are collected from Inner Mongolia, China. Totally 230 Lactobacillus strains are isolated 12 and screened by acid-, biletolerance, lactose decomposition and acid production ability. The cholesterol lowering abilities and adhesiveness on Caco-2 are evaluated. The top 2 strains are identified as Lactobacillus plantarum. These 2 strains are prepared as the starter cultures in milk 15 fermentation. The development technology of starter cultures is studied.


INTRODUCTION
The research of new functional foods that isolate and identify new strains of lactic acid bacteria from traditional dairy products may lead to novel bacterial strains with functional characteristics and revealing taxonomic characteristics. It is known that camel has a long lactation period (more than 15 months). Camel milk is sticky milk with normal smell. It contains large contents of dry matter, high milk fat rate and small milk fat ball. 24 As a good source of amino acids, camel milk is an ideal milk source. The most interesting characteristic is that the natural acidity of camel milk is at an average of 22°T. It is 16-18°T in cow milk and 6.5°T in mares' milk. Camel milk can be preserved for a long time after processing or 27 acidification without corruption, while cow milk cannot [1]. Traditional fermented camel milk, due to its rarity and high nutritional value, provides good conditions for microbial research [2,3]. Khoormog is a unique Mongolian fermented camel milk with high nutritional value, unique flavor, 30 and form many probiotic groups with good passage under the spontaneously fermentation [4]. As a rich resource of lactic acid bacteria, the fermented camel milk Khoormog was studied in the present study. 33 Current foods are rich in lipids. Cholesterol is an organic molecule, a type of lipid. Exogenous intake of cholesterol is mainly obtained from the diet. Thus, a higher intake from food leads to a net decrease in endogenous production and the increase of cholesterol in blood. Higher cholesterol in 36 blood is associated with risks of heart disease [5][6][7]. Therefore, there has been considerable research on lowering cholesterol through a healthy and reasonable diet, especially through the use of lactic acid bacteria [8]. There are two theories support lactic acid bacteria to lower cholesterol. One theory 39 is that some in vitro studies showed the cells of lactic acid bacteria strains can absorb cholesterol when grow in the high cholesterol medium containing bile salts under anaerobic conditions, and reduce the cholesterol in the medium [9]. The second theory derives from in vitro studies which ORH-801, mobile phase 5 mMol/L of H2SO4 with flow rate 0.8 mL/min, UV detector at 210 nm and column temperature 35°C. The viable count of lactic acid bacteria in each sample was counted according to the method of 72 Kanno et al. [14] with some modification. 1 g of each sample and 9 mL of phosphate buffered saline solution (PBS, Biosharp, China, pH 7.2) containing 1 g/L agar were homogenized. Ten-fold serial dilutions (from 10 -1 to 10 -5 ) of each sample were prepared. 50 µl of each dilution were directly 75 inoculated on de Man Rogosa Sharpe plates (MRS agar, Oxoid, UK). The MRS plates were incubated at 37°C and 25°C under anaerobic conditions using the AnaeroPack system (Mitsubishi Gas Chemical, Japan) respectively. Viable count was counted and calculated after 48 h of incubation 78 as log colony forming units (CFU) per gram of sample.

Screening of acid-and bile-tolerance, lactose utilization and acid production ability:
Colonies grown on the MRS agar plates were isolated and purified after the viable count calculation. The yellow round shape colonies around with the clear zone were randomly selected. Ann average of 9-84 10 colonies were isolated from each sample incubated at two temperatures. A total of 230 colonies were isolated [13]. Gram positive, catalase negative and rod strains were isolated. The lactic acid bacteria isolates were screened by acid-, bile-tolerance and lactose utilization tests 87 by in vitro. The acid tolerance of each isolates were inoculated into 5 mL MRS broth which adjusted to pH 3.0. After 24 h incubation at 37°C, absorbance at OD600 was measured, ∆A≥0.5 was chosen [15]. The bile tolerance of each isolate was determined by inoculation into 5 mL MRS broth 90 containing 10 g/L bile (Oxgall, Neogen, USA). Absorbance at OD620 was measured. A decrease in pH indicates the lactose utilization ability because of acidic fermentation products. Each isolate was inoculated into in Gifu Anearobic Medium (GAM) semi-solid medium (Nissui, Japan) containing 93 1% (w/v) lactose. BTB-MR reagent (0.1 g bromothymol blue, 0.2 g methyl red, 300 mL ethanol, and 200 mL distilled water) was used [16]. According to acid-, bile-tolerance and lactose utilization tests, 34 lactic acid bacteria isolates were selected for acid production ability testing. 96 Acid production capacity was evaluated according to the method of Jicheng [17] with slight modification. Skimmed milk powder (Members Mark, New Zealand) was 10% (w/v) hydrated and heated at 95°C for 5 min (pH 6.6) [18]. After cooling to 37°C, lactic acid bacteria isolates were 99 inoculated. The pH was recorded every 2 hours until the end of the fermentation (pH 4.6). The formation of milk curd was observed and a viable count undertaken. Based on sample origin, fermented milk observation and acid production ability, the top 9 lactic acid bacteria isolates were 102 then selected for the subsequent experiments. 16S rDNA gene sequence analysis: The top 8 screened lactic acid bacteria isolates were identified using 16S rDNA gene sequence analysis. TIANamp bacteria DNA kit (TIANGEN, China) was used 105 for the genomic DNA extraction. PCR amplification was performed according to the previous study by Jie [19]. Forward primer 27F (5'-AGA GTT TGA TCC TGG CTC AG -3') and reverse primer 1541R (5'-AAG GAG GTG ATC CAG CCG CA -3') was used [20]. PCR amplification program 108 was as follow: 94°C for 4 min, followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 2 min, and finally 72°C for 5 min. DNA sequencing was performed by Shanghai Sangni Biotechnology Co., Ltd. The sequences were analyzed through the BLASTn database at GenBank 111 (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Evaluation of cholesterol lowering ability by in vitro tests:
The evaluation of cholesterol lowering ability of the 8 isolates was studied in the College of Animal Science, Inner Mongolia Agricultural concentrated and freeze dried. Skim milk powder was 10% (w/v) hydrated and heat treated at 95°C for 5 min and then cooled to 43°C. The freeze dried strain powder was dissolved into the skim milk at 3% (w/v). Then incubated at 43°C, until it reached pH 4.6. Production starter culture was prepared 153 after 3 times of activation. Production starter culture activity was inspected [24] and the acidity recorded every 2 hours of fermentation until pH 4.6. The initial acidity of skim milk was pH 6.7, titratable acidity 18°T. The Resazurin reduction was tested [25], 1 mL of each production culture 156 and 0.005% (v/v) of resazurin solution was dissolved into 9 mL skim milk. Incubation at 36.7°C for 35 min.

Determination of optimal temperature and inoculum in milk fermentation tests: The 2 Lb.
159 plantarum strains were prepared as the starter cultures and fermented in cow's milk according to Gu [25] with some modification. Ultrahigh temperature (UHT) sterilized milk (whole milk, MLEKOVITA, Poland) was used. To determine the optimal temperature, the starter cultures of the centrifuged at 5000 r/min for 10 min. Took 10 mL supernatant into the special cup of the Taste Sensing System using a pipette gun. Diluted with triply distilled water and stood for 1 h. The tastes 171 of sweetness, sourness, bitterness, astringency and umami were set and determined. Sensory evaluation was performed by seven trained researchers after milk fermentation according to Chinese standard RHB 103-2004 [26]. Briefly, the fermented milk samples were filled into 50 mL beakers 174 and the color and texture was observed under the light. Researchers gargled with warm water after smelled the yogurt smell, then tasted. One hundred points system was used to evaluate the fermented milk. The evaluated sensory items included color (0-10 points), taste and smell (0-40 points), texture 177 (0-50 points). The highest and the lowest points of evaluation were removed and the total points were calculated.
To determine the optimal inoculum, cow's milk was prepared as mentioned above. The starter 180 cultures of the 2 isolates were inoculated at 1, 3, 5, 8, and 10 g/100 g of inoculum respectively. The inoculated milk was incubated at 43°C. The acidity, viable count, water holding capacity, tastes and sensory evaluation was recorded. 183 All the above experiments were replicated three times in comparison with the standard Lb. plantarum strain P8. The analysis of variance (ANOVA) was performed using IBM SPSS statistics 25 programme software (Showed in Appendix).

186
Central composite design of optimal condition in milk fermentation: According to the single factor experiment of determination of optimal temperature and inoculum, the optimal condition in milk fermentation of the 3 starter cultures was obtained through response surface experiment design. 189 Central composite design was performed using the Design-Expert 12 software. Midland of Inner Mongolia, ranged from 3.8 to 5.5 Log 10 CFU/g, in the other two groups ranged from 6.5 to 8.9 Log 10 CFU/g. In previous study by Shuangquan [29], the viable count of dairy products in Inner Mongolia ranged from 5.3 to 8.7 log CFU/g. Thus, three samples (T1, T2, and T3) 204 collected from the group of the Midland of Inner Mongolia could be considered that it still in the early stage of fermentation. The samples in the other groups could be considered that it had entered late fermentation, and stored for a long time. Including the samples (T5 to T13) collected from the 207 group of the Midwest and Western Inner Mongolia, showed high bacterial activities. Screening and identification: Lactic acid bacteria were isolated from twenty three Khoormog samples according to their individual characteristics. Colonies grown on the MRS agar plates were purified and isolated. Among 230 gram positive and catalase negative rod strains, 172 grew in MRS 213 broth adjusted to pH 3.0. Among the acid tolerance isolates, 69 produced acid from lactose. Among the acid tolerance and lactose decomposed isolates, 34 grew in the 10 g/L bile condition. The 34 acid-and bile-tolerant and lactose decomposed isolates were tested by their acid production ability. 216 Based on the sample origin, fermented milk observation and acid production ability, the top 8 isolates were selected. The acid production ability of the 8 isolates was shown in Table 3. The fermentation period ranged from 6.5 h to 7 h at the end of the fermentation (pH 4.6). The viable 219 count ranged from 7.1 to 7.5 Log 10 CFU/g. The identification by 16S rDNA gene sequence analysis is shown in  [30]. 225 Before any strains of functional bacteria can be used as a food adjunct, they must be able to survive in the acidic conditions in stomach and resist bile acids in order to be effective on the host [31]. Once ingested, the bacteria is exposed to the harsh digestive processes for more than 90 minutes 228 before being released from the stomach into the intestine [32]. Acid tolerance is often a crucial factor considering that pH of stomach could be as low as 2.5 in fasting subjects [19]. In addition, when probiotics pass through the upper intestinal tract, they are subjected to varying bile types and 231 concentrations that may decrease their viability [31]. In order to simulate the human gastrointestinal environment, the acid-(pH 3.0) and bile-(10 g/L) tolerance tests were conducted in vitro. The lactose (1% w/v) utilization test was aimed at the lactose intolerance, which is mostly reported in 234 Asia and Africa [13]. Lactose intolerance is a congenital hereditary disease in which the body does not produce lactase to utilize lactose. Flora decomposes lactose in the intestinal tract into lactic acid to destroy the alkaline environment of the intestine. Intestines have to secrete a large amount of 237 alkaline digestive solution to neutralize the lactic acid which causes diarrhea [33]. Among the 230 lactic acid bacteria isolates, 34 acid-and bile-tolerance and lactose decomposed isolates were screened and their acid production ability was tested. Based on the sample origin, fermented milk 240 observation and acid production ability, the top 8 isolates were selected for the further functional experiment.  conditions, and function in vivo to exert a hypocholesterolemic effect [22]. Based on the cholesterol lowering ability, the Lb. plantarum strains AM2-6, BM2-5 and BM4-2 were selected as the pending strains for further fermentation experiment. 258 Deconjugation of sodium taurocholate of the 8 isolates ranged from 1.12±0.07 μmol/mL to 2.04±0.21 μmol/mL (    Production starter culture activity of AM2-6, BM2-5 and P8 was recorded ( Table 6). The starter cultures of AM2-6, BM2-5 and P8 showed an acid production ability during 8 h fermentation. The fermentation period of starter cultures of AM2-6, BM2-5 and P8 were about 7 h (pH 4.6). Starter 321 cultures of AM2-6 and BM2-5 showed a higher acid production ability than the known P8 during the 4 h to 6 h of fermentation. Among them, AM2-6 showed stronger acid production ability in a short time (P<0.05). This is consistent with the previous studies by Pan et al. [38]. In a previous 324 study by Song [39], the fermentation period of Lb. plantarum starter cultures was about 8 h to 9.3 h. Resazurin solution of the 3 starter cultures were completely faded indicating that the 3 of Lb. plantarum starter cultures kept high activity and could be used in the milk fermentation.  Determination of optimal temperature in milk fermentation tests: Lb. plantarum starter cultures of AM2-6, BM2-5 and P8 were provided for cow's milk fermentation. To determination of optimal fermentation temperature, 37, 39, 41, 43, and 45°C were trialed. The inoculum used was 3 g/100 g 333 milk. A pH of 4.6 was taken to be the end of fermentation. The optimal fermentation temperature for the fermented milk should meet the following conditions. Viable count should over 6 Log 10 CFU/g [40]. The curd texture is moderate with milky white color, with no whey separated. Keeping 336 high water holding capacity, good tastes and high sensory score are also important. The taste determination by Taste Sensing System of the fermented milk is shown in Fig 3. (A: BM2-5, B: AM2-6, C: P8). The tastes were set as sweetness, sourness, bitterness, astringency and umami. 339 The tastes of sourness, bitterness and umami showed significant differences (P<0.05), sweetness and astringency didn't show significant differences (P>0.05). It indicated that the fermentation temperature had an effect on the taste of the fermented milk. The taste of sourness showed the most 342 significant differences in the three fermented milk, it was followed by bitterness and umami. In general, the sourness increased as the fermentation temperature increased whilst bitterness and umami decreased as the fermentation temperature increased. It indicated that the increase in 345 sourness can not only reduce bitterness in the fermented milk, but also reduce umami taste. The fermentation period, viable count, water holding capacity and sensory evaluation of the fermented milk in different fermentation temperature is shown in Table 7. The fermentation periods 348 of the three fermented milk decreased as the fermentation temperature increased. According to the viable count, water holding capacity and sensory evaluation of the fermented milk, the optimal temperature for the fermentation of milk was 43°C. At 43°C the fermentation period of the 3 starter 351 cultures was about 7 to 7.5 h. In the case of AM2-6, viable count of the fermented milk was 7.3 Log 10 CFU/g, fermentation period was 7 h. The curd texture was moderate, milky white color, and no whey separated. Water holding capacity was 66.2%. Sensory score was 91. In the case of BM2-5, 354 viable count was 7.0 Log10 CFU/g, fermentation period was 7 h 10 min, water holding capacity was 65.7% and sensory score was 90. In the case of P8, viable count was 6.9 Log 10 CFU/g, fermentation period was 7 h 20 min, water holding capacity was 61.6%, sensory score was 90. 357 In major dairy factories, the fermentation temperature of the fermented milk ranges from 37 to 43°C [41]. In other words, the differences of the fermentation temperature depend on the differences of the starter cultures. Therefore, the optimal fermentation temperature of the 2 Lb. plantarum strains 360 AM2-6 and BM2-5 were determined for a better understanding of the potential health benefits. The fermentation temperatures trialed in the present study were 37, 39, 41, 43 and 45°C. According to the previous study by Hao [42], both high and low temperature fermentation reduced the water 363 holding capacity of the fermented milk, a fermentation temperature between 36°C and 44°C had no significant effects on the water holding capacity of fermented milk. In the present study, the water holding capacity of fermented milk showed significant differences during fermentation between 366 37°C and 45°C (P<0.05). Both AM2-6 and BM2-5 showed a high water holding capacity under Mongolian Journal of Chemistry xx of xx 43°C of fermentation, and significantly higher than the known P8 (P<0.05). In general, the fermentation periods decreased as the temperature increased. Viable count was over 6 Log 10 CFU/g 369 in all of the fermented milk. Combined with the tastes determination by Taste Sensing System and sensory evaluation by seven trained researchers, the fermentation temperature has certain effects on the fermented milk. The taste of sourness increased, the bitterness and umami decreased as the 372 fermentation temperature increased. The differences of sourness supposed to related to the growth temperature of Lb. plantarum strains. The differences of bitterness and umami may also have related to free amino acids in the fermented milk. For example, glutamic acid (Glu) has an umami taste and 375 glycine (Gly) has a sweet taste [25]. The fermented milk produced under 43°C by both AM2-6 and BM2-5 showed a good taste.

Determination of optimal inoculum in milk fermentation tests:
To determine the optimal inoculum 384 of the starter cultures 1, 3, 5, 8, and 10 g/100 g of inoculum respectively. The inoculated milk was incubated at 43°C with pH 4.6 indicating the end of the fermentation. The optimal inoculum of the fermented milk should meet the conditions as mentioned above. 387 The taste determination in different inoculum is shown in Fig 4. (A: BM2-5, B: AM2-6, C: P8). The tastes of sourness, bitterness and umami showed significant differences (P<0.05), sweetness and astringency didn't show significant differences (P>0.05). The fermentation period, viable count, 390 water holding capacity and sensory evaluation of the fermented milk in different inoculum is shown in Table 8. When increased or decreased the inoculum of the starter culture, the fermentation period was prolonged, and sensory evaluation was decreased. Combined the viable count, water holding 393 capacity and sensory evaluation of the fermented milk shown in Table 8, the optimal inoculum of the starter cultures AM2-6 and BM2-5 was 3 g/100 g, and P8 was 5 g/100 g.  Central composite design of optimal condition in milk fermentation: In central composite design, two numeric factors and three responses were set. The two numeric factors were fermentation temperature and the dose of inoculum in each fermented milk, the three responses were fermentation 402 period, viable count and water holding capacity. The desirable optimal condition in milk fermentation according to the central composite design is shown in Fig 5 (A: BM2-5, B: AM2-6, C: P8). In the case of AM2-6, the optimal fermentation temperature is 43.9°C, the optimal inoculum is 405 3.9 g/100 g. In the case of BM2-5, the optimal fermentation temperature is 43.3°C, the optimal inoculum is 3.6 g/100 g. In the case of P8, the optimal fermentation temperature is 43.6°C, the optimal inoculum is 5.4 g/100 g. The results of response surface design by central composite design 408 were consistent with the single factor experiments. Therefore, combined with the results of single factor experiment and response surface design, the optimal fermentation conditions of the Lb. plantarum starter cultures AM2-6, BM2-5 and P8 were as follows. The optimal fermentation 411 temperature was 43°C for the three Lb. plantarum starter cultures. The optimal inoculum of AM2-6 and BM2-5 was 3 g/100 g, and P8 was 5 g/100 g. adhesion of lactic acid bacteria in dairy products. Therefore, the Lb. plantarum strains AM2-6 and BM2-5 could be used as probiotics in functional foods to reduce serum cholesterol. These two strains were selected as the typical strains for the preparation technology of starter cultures in 432 comparison with the known Lb. plantarum strain P8 in milk fermentation. The optimal fermentation temperature and optimal inoculum of development technology of starter cultures were studied by single factor experiments and response surface experiment design. As mentioned above, not every 435 Lb. plantarum strains have the ability to ferment in milk. The resource of isolation is important. In this study, AM2-6 and BM2-5 could be used for the fermented milk production with high fermentation ability. Even compared to the known P8, AM2-6 could exceed P8 in cow's milk 438 fermentation.