Source: LOUISIANA STATE UNIVERSITY submitted to NRP
INFLUENCE OF MILD PROCESSING CONDITIONS OF VARIOUS PROCESSES ON ENHANCING THE PROBIOTIC CHARACTERISTICS OF HEALTH BENEFICIAL BACTERIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0224571
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Dec 1, 2010
Project End Date
Dec 1, 2015
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
School of Animal Science
Non Technical Summary
Lactobacillus bulgaricus and Streptococcus thermophilus are two required micro organisms in yogurt manufacture according to the definition of yogurt (Code of federal regulations (21 CFR131.200), 2009). Lactobacillus acidophilus is a probiotic bacterium widely used as an adjunct culture in approximately 80% of the yogurts being manufactured in the USA (Hutkins, 2006). Heller (2006) reported 3% increase in sales of cultured dairy products in USA from 2004 to 2005 making $9.7 billion in the year 2005, of which 50% sales ($4.9 billion) were contributed by yogurt. Consumption of yogurt was enhanced mainly because of its nutritional value and the beneficial health effects of yogurt bacterial cultures. Dairy Facts (2009) reported 3.44 % increase in sales of yogurt from 2007 to 2008. A "live and active cultures" seal has been developed by the National Yogurt Association to help yogurt consumers identify that the yogurt contains "live and active cultures" to enable consumers make sure that they are getting the most from their yogurt. In order for manufacturers to carry the "live and active cultures" seal on their yogurt label, the refrigerated yogurt must contain at least 100 million culture organisms / gram at the time of manufacture (National Yogurt Association, 2009). Viability of yogurt cultures and probiotic bacteria is affected by inhibitory substances such as lactic acid produced during production and cold storage. During production of yogurt, yogurt bacteria and probiotic bacteria produce organic acids (Shah 2000). Nogueira et al., (1998) reported a 2 log reduction in counts of L. bulgaricus and a one log reduction in counts of S. thermophilus over 4 weeks of yogurt storage. Simova et al., (2008) reported a two log reduction in counts of both L. bulgaricus and S. thermophilus in yogurt over 14 days (2 weeks) of storage. Yuceer et al., (2000) reported a 1.5 log reduction in counts of L. bulgaricus and S. thermophilus in yogurt over a 90 days storage period. The pH of the product needs to be 4.5 or lower to meet the legal requirement and produce good quality yogurt. Shah (2000) reported that pH was the most crucial factor for the survival of Lactobacillus acidophilus and that if the pH of yogurt dropped below 4.4 at the time of fermentation it resulted in a 3 to 4 log cycle decrease in Lactobacillus acidophilus counts. Several studies reported yogurt cultures and L. acidophilus as probiotics / health beneficial bacteria. Probiotic bacteria upon being ingested, have to survive the acid and bile conditions before reaching and establishing in the lower gastrointestinal tract. Improving the probiotic characteristics such as acid tolerance and bile tolerance of dairy culture and probiotic bacteria would be advantageous to its consumers and the fermented dairy foods industry. Outcomes / impacts: We need processing to manufacture food and the influence of various processes on probiotics is not know which this study will address. If the results are encouraging this could lead to new methods of processing foods in future for increased health benefits.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5013430110030%
5013450110010%
5013460110010%
5023430110030%
5023450110010%
5023460110010%
Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
3430 - Cheese; 3450 - Milk; 3460 - Ice cream;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
GOALS: Overall goal is to improve the beneficial characteristics of health beneficial bacteria. The specific goals are to improve the acid tolerance, bile tolerance and growth of probiotic bacteria. OBJECTIVES are 1) To determine the effect of low homogenization pressures such as 0, 1.725, 3.447, 5.175, 6.895, 8.625, 10.342, 12.065 and 13.790 MPa on characteristics such as acid tolerance, bile tolerance, protease activity and growth of bacteria such as Lactobacillus delbrueckii ssp bulgaricus, Streptococcus salivarius ssp thermophilus and Lactobacillus acidophilus. 2)To elucidate the influence of mild sonication conditions such as amplitudes of 13.02 micrometers (um) (21%), 16.74 um (27%), 20.64 um (33%) and 24.18 um (39%) at processing temperatures such as 4, 22 and 40C on characteristics such as growth, bile tolerance, acid tolerance, and protease activity of bacteria such as Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus. EXPECTED OUTPUTS are presentations at scientific meetings and publications in refereed journals.
Project Methods
Homogenization: One gallon of skim milk will be autoclaved and cooled to 4C. This sterilized milk will be individually inoculated with 0.1% (v/v) with bacteria such as Lactobacillus delbrueckii ssp bulgaricus, Streptococcus thermophilus and Lactobacillus acidophilus. The control sample will be treated at 0 MPa for 1 minute (5 passes) in a sterilized homogenizer and the homogenized samples will be those subjected to homogenization pressures such as 1.725, 3.447, 5.175, 6.895, 8.625, 10.342, 12.065 and 13.790 MPa for 1 minute (5 passes). The control and homogenized samples will be tested for growth, bile tolerance, acid tolerance and protease activity. Growth will be determined by plating the broth inoculated with the homogenized cultures every hour for 10 hours while the broth is maintained in incubation conditions. Bile tolerance of the strains will be tested by adding the bile acids to the broth, inoculated with the homogenized cultures and plating every hour for 10 hours similar to the growth test. Acid tolerance of the three strains will be tested by inoculating the homogenized cultures in the acidified broth and plating for every 20 minutes up to 2 hours. Protease activity of the homogenized strains will be determined by measuring optical density value using spectrophotometer at 0, 12 and 24 hours of incubation of the samples. Three replications will be conducted. Sonication : A sterilized sonicator probe will be used to deliver various amplitudes such as 13.02 micrometers (um) (21%), 16.74 um (27%), 20.64 um (33%) and 24.18 um (39%), will be used to treat cultures such as Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarus spp. thermophillus and Lactobacillus acidophilus in sterile peptone water at temperatures such as 4 C, 22 C and 40 C. All treatments will have constant frequency (20 kHz) and energy (1500 Joules). Control samples will be cultures in sterile peptone water at same temperatures but not receiving any sonication. The control and sonicated samples will be tested for growth, bile tolerance, acid tolerance and protease activity at time periods described above. Three replications will be conducted. Characteristics: Growth of Lactobacillus delbrueckii ssp bulgaricus, Streptococcus thermophilus and Lactobacillus acidophilus will be determined by the method proposed by (Simov et al., 2006). The acid tolerance and bile tolerance of the three cultures will be determined by the method proposed by Pereira and Gibson (2002). The protease activity will be determined according to method proposed by Oberg et al. (1991) Statistical analyses: Data will be analyzed by ANOVA using the Proc mixed linear model procedure of SAS (SAS Institute Inc. 2001). Means will be separated using the least significant difference test. Significant differences will be determined at α = 0.05.

Progress 12/01/10 to 12/01/15

Outputs
Target Audience:Dairy and Food industries, academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate and undergraduate students were trained on these projects. Additional interesting hypotheses were developed. How have the results been disseminated to communities of interest?Results have been disseminated through peer reviewed publications and presentations at national level scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Yogurt mixes were made by incorporating the starches individually into skim milk. The yogurt mixes were pasteurized at 65.5°C for 30 min, cooled to 40°C, inoculated with freshly thawed Streptococcus thermophilus (ST-M5) (3.1E+10 cfu/g, 1ml) and Lactobacillus bulgaricus (LB-12) (3E+10 cfu/g, 1ml) per 3.785L (1 gallon), then incubated at 40°C until they reached a pH 4.5, and held at 4°C overnight. Blueberry puree (20% w/w) was incorporated into the yogurt the following day, and amylopectin starch (15 g, control, AMIOCA® corn starch) or HAM-RS2 (15g, HI-MAIZE® 260, resistant starch) per 237ml serving was added to the yogurt. A high performance liquid chromatography (HPLC) peak was detected in the HAM-RS2 sample, and RS accounted for 38.2% of the sample. One hundred and ten children, of 7-8 (n=91) or 13-14 (n=19) years of age, scored the sensory qualities of a yogurt supplemented with either HAM-RS2 or an amylopectin starch. The amylopectin starch yogurt was preferred to the HAM-RS2-enriched yogurt by 7-8 year old panelists (P<0.0001). Appearance, taste, and sandiness scores given by 13- to 14-year-old panelists were more favorable for the amylopectin starch yogurt than for HAM-RS2-enriched yogurt (P<0.05). HAM-RS2 supplementation resulted in acceptable (≥6 on a 1-9 scale) sensory and hedonic ratings of the yogurt in 74% of subjects. HAM-RS2 was not modified by pasteurization of the yogurt mix and may be a palatable way to increase fiber intake and stimulate colonic fermentation in adolescents. Ice creams were manufactured with 0, 1.5, 3.0 and 4.5 g colostrum per pint (473 mL) of ice cream. Ice cream manufacture was replicated three times. Colostrum incorporation increased aerobic counts andapparent viscosity but decreased meltdown resulting in slower melting of the ice creams and had no influence on flavor. In an attempt to make healthier ice creams, colostrum incorporation can be recommended in ice cream manufacture. The cheese cracker treatments consisted of 3 different salt sizes (regular, microsalt and submicrosalt) and 3 different concentrations (2, 1.5 and 1%). The 9 (3 sizes x 3 concentrations) different cheese cracker treatments were tested for salt concentration and sodium content at week 1. Water activity, yeast and mold counts, texture-fracturability, color, and consumer test were determined at week 1 and 4 months of storage. Completely Randomized Design (CRD) with repeated measures was used for water activity, yeast and mold counts, texture-fracturability, and color. A Balanced Incomplete Block Design was used to conduct the consumer analysis of cheese crackers for submicrosalt (2, 1.5 and 1%), microsalt (2, 1.5 and1%) and regular 2% (control as used by industry) using 476 participants. Submicrosalt treatments (2, 1.5 and 1%) had a positive effect in yeast reduction at 4 months compared to regular salt (2, 1.5 and 1%). There was no mold growth in all treatments at all times. The L*, b*, C* and h* values in all treatments increased significantly (P<0.05) from 1 week to 4 months. At 4 months, submicrosalt treatments (2, 1.5 and 1%) resulted in having significantly (P<0.05) more preferred saltiness scores compared to control (regular 2%). At 4 months, submicrosalt (1.5 and 2%) showed significantly (P<0.05) more preferred just about right saltiness scores compared to control. The reduction of 25 and 50% salt content in cheese cracker through use of submicro particulated salt maintained low counts in yeasts; no counts in molds; and did not adversely influence sensory color, aroma, crunchiness, overall liking and acceptability scores, which were the same compared to control and microsalt treatments (2, 1.5 and 1%). Reduction in salt / sodium chloride particle size 1000 fold from regular salt to submicrosalt increased saltiness of surface salted cheese crackers.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Vargas L., Olson, D. and Aryana K.J. 2015. Whey protein isolate improves acid tolerance and bile tolerances of Streptococcus thermophilus ST-M5 and Lactobacillus delbrueckii ssp. bulgaricus LB-12. Journal of Dairy Science 98 (4): 2215-2221.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Moncada, M., Astete, C., Sabliov, C., Olson D., Boeneke, C. and Aryana K.J. 2015. Nano spray dried sodium chloride and its effects on the microbiological and sensory characteristics of surface salted cheese crackers. Journal of Dairy Science 98 (9): 5946-5954.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Aryana K. J., Greenway Frank L., Dhurandhar Nikhil V., Tulley Richard, Finley John. W., Keenan Michael.J., Martin Roy.J., Pelkman Christine, Danuta Janik, Olson Douglas and Zheng Jolene. 2015. A resistant-starch enriched yogurt: fermentability, sensory characteristics and a pilot study in children. F1000Research. 4:139.
  • Type: Other Status: Accepted Year Published: 2015 Citation: Aryana, K.J. and Vargas L., 2015. Using microorganisms in the manufacture of dairy foods. Louisiana Agriculture, Fall 2015. Accepted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ernesto E., K.J. Aryana (2015) Influence of osmotic adaptation and lactose deprivation on the salt tolerance of Lactococcus lactis. ADSA annual meeting July 12-16, Orlando, FL. J. Dairy Sci. Vol 98. Suppl 2: 362.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Osorio I and K. J. Aryana (2015). Heat tolerance of Lactoccocus lactis with prior subjection to mild heat stress. ADSA annual meeting July 12-16, Orlando, FL. J. Dairy Sci. Vol 98. Suppl 2: 361.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Duferene A., D. Park, D. Olson., K. J. Aryana (2015). Can probiotic bacteria survive in a beverage made from acid whey from Greek yogurt? ADSA annual meeting July 12-16, Orlando, FL. J. Dairy Sci. Vol 98. Suppl 2: 363.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ernesto E., K.J. Aryana (2015). Effect of ultraviolet light exposure and mild heat shock on the salt tolerance of Lactococcus lactis. ADSA annual meeting July 12-16, Orlando, FL. J. Dairy Sci. Vol 98. Suppl 2: 361.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2015 Citation: Emilio E. 2015. Salt tolerance of Lactococcus lactis R-604 as influenced by exposure to various stress conditions. Thesis submitted to Louisiana State University. Major Professor, Aryana K.J.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: OBrien, K.V., Boeneke, C. A., Stewart, L.K. and Forney, L. W. Prinyawiwatkul and K.J. Aryana. 2015. The effects of post-exercise consumption of a kefir beverage on performance and recovery during intensive endurance training. Journal of Dairy Science. 98(11):7446-7449.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Mouton E. and Aryana K.J. 2015. Influence of colostrum on the characteristics of ice cream. Food and Nutrition Sciences. 6: 480-484.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Scientific community, Dairy / Food Industry Changes/Problems: No changes / problems were experienced. What opportunities for training and professional development has the project provided? One Masters thesis, two articles in Journal of Dairy Science, eight presentations at national level scientific meetings, andtraining of two undergraduate students on research techniques. How have the results been disseminated to communities of interest? Through the 8 presentations this year at a national level scientific meeting with published online abstracts and refereed journal articles. What do you plan to do during the next reporting period to accomplish the goals? Continue researching on enhancing desirable / probiotic characteristics.

Impacts
What was accomplished under these goals? There is a pronounced public awareness about herbal remedies. Earlier work has shown the influence of spice extracts but the influence of pure spice juice on yogurt culture bacteria is not known. Hypothesis was different spice juices have different influences on probiotic characteristics and yogurt attributes. The following variables were investigated 1) To determine the influence of 1% (v/v) of garlic, ginger, onion and turmeric juice on the growth of Streptococcus thermophilus ST-M5, Lactobacillus bulgaricus LB-12 and Lactobacillus acidophilus LA-K. 2) To elucidate the influence of 1% (v/v) of garlic, ginger, onion and turmeric juice on acid tolerance, bile tolerance and protease activity of S. thermophilus, L. bulgaricus and L. acidophilus. 3) To manufacture a spicy probiotic yogurt and to elucidate the influence of 0.05% (v/v) of garlic, ginger, onion and turmeric juice on the growth of S. thermophilus, L. bulgaricus and L. acidophilus in yogurt. 4) To elucidate the influence of 0.05% (v/v) of garlic, ginger, onion and turmeric juice on the physico-chemical characteristics (pH, titratable acidity, coliform bacterial growth, color and apparent viscosity) of spicy probiotic yogurt. 5) To study the influence of 0.05% (v/v) of garlic, ginger, onion and turmeric juice on sensory characteristics of spicy probiotic yogurt and to determine the consumer acceptability of spicy probiotic yogurt. Freshly thawed Streptococcus thermophilus ST-M5, Lactobacillus bulgaricus LB-12 and Lactobacillus acidophilus LA-K culture were suspended in sterilized 0.1% peptone water and 1% (v/v) of freshly extracted spice juice individually of garlic, ginger, onion or turmeric. The four spice juice treatments were performed in a random manner. The control was the sample in which no spice juice was added. Growth was determined by plating the control and the treated samples every 12 hours for 60 hours of incubation for S. thermophilus and every two hours for 8 hours of incubation for the Lactobacilli. Bile tolerance was determined by plating the control and the treated samples every hour for 5 hours of incubation. Acid tolerance was determined by plating the control and the treated samples every hour for 2 hours of incubation. Protease activity was determined by measuring the optical density (absorbance) at 0, 12 and 24 hours of incubation. A spicy probiotic yogurt was manufactured with 0.05% of the juice, individually of the 4 spices. Physico-chemical characteristics, namely pH, titratable acidity, color, apparent viscosity and coliform bacterial growth, as well as growth of the three organisms in yogurt were measured weekly for a period of 35 days. Three replications were conducted. Experimental design was a randomized block design (RBD) with repeated measures. Results indicated that the juice in a concentration of 1% (v/v) of garlic, ginger, onion and turmeric did not have an inhibitory effect on the growth of Streptococcus thermophilus ST-M5, Lactobacillus bulgaricus LB-12 and Lactobacillus acidophilus LAK. Turmeric showed the lowest reduction in viable counts for S. thermophilus and L. acidophilus, while L. bulgaricus grew best in the presence of onion, when compared to control. All four spices decreased the acid and the bile tolerance of S. thermophilus, but still with viable counts. Garlic, ginger and onion improved the bile tolerance of L. bulgaricus, while turmeric decreased it when compared to control, but still with viable counts. Ginger, turmeric and onion had the best overall influence on the protease activity of S. thermophilus, when compared to control. Turmeric improved the protease activity of L. bulgaricus and alongside with ginger, it also improved the protease activity of L. acidophilus, when compared to control. The pH of the spicy blueberry yogurt was lower in the presence of all four spices when compared to control, while the titratable acidity was higher in the presence of garlic, ginger and turmeric, when compared to control. At day 1, coliform counts of turmeric were significantly higher compared to the rest of the spices and the control, while at all other storage time points there were no coliform counts. Streptococcus thermophilus obtained from the spicy blueberry yogurt grew better in presence of garlic and ginger when compared to control, while L. bulgaricus (from the spicy blueberry yogurt) grew better in the presence of onion and turmeric when compared to control. The counts of Lactobacillus acidophilus in the blueberry yogurt with all spices were about half a log lower when compared to control. The apparent viscosity of the spicy blueberry yogurt was particularly higher in the presence of onion and ginger, when compared to control. The control yogurt obtained 80% acceptance and the ginger yogurt obtained 84% acceptance. Ginger yogurt obtained the highest intent of purchase. Ginger can be used in yogurts for direct consumption while all 4 spices can have potential for a new product line of yogurts for cooking, marinating and dips enabling potential for health benefits from some spices and some bacteria. For manufacture of commercial yogurt powder, yogurt has to go through a drying process which substantially lowers the yogurt culture counts, so the potential health benefits of the yogurt culture bacteria are reduced. Also, upon reconstitution commercial yogurt powder does not taste like yogurt and has an off-flavor. Purpose of this study was to determine whether reconstituted YCMP mix would have higher culture bacterial counts and better physico-chemical and sensory characteristics than reconstituted CYP currently available. This study consisted of CYP reconstituted yogurt as the control and YCMP mix reconstituted yogurt as the treatment. For the first and second objectives, microbial and physico-chemical characteristics of the reconstituted yogurts were analyzed daily for the first wk and then weekly for a period of 8 wkafter reconstitution. Three replications were conducted. For the third objective, consumer tests of sensory characteristics of reconstituted yogurts from YCMP mix, YCMPA mix and CYP were conducted with 100 panelists. Results showed that YCMP mix reconstituted yogurt had 5 log cfu/ml higher counts of Streptococcus thermophilus compared to the control (CYP reconstituted yogurt) at 56 days. Also, Lactobacillus bulgaricus counts of YCMP mix reconstituted yogurt at 28 days was 6.55 log cfu/ml and at 56 days was 5.35 log cfu/ml while the CYP reconstituted yogurt at 28 days onwards had no detectable counts. CYP reconstituted yogurt cannot meet the recommended yogurt culture bacteria concentration of 6-8 log cfu/g which can be met by YCMP mix reconstituted yogurt. No coliform was observed at 56 days for both CYP and YCMP mix reconstituted yogurts. Apparent viscosity of YCMP mix reconstituted yogurt was significantly higher than CYP reconstituted yogurt. YCMP mix reconstituted yogurt had significantly higher pH but significantly lower TA than CYP reconstituted yogurt. YCMP mix reconstituted yogurt had significantly higher appearance, color, aroma, taste, thickness, powderiness and overall liking scores compared to CYP reconstituted yogurt. Consumer acceptability of YCMP mix reconstituted yogurt (54%) was more than twice that of CYP reconstituted yogurt (22%). YCMP mix reconstituted yogurt had a markedly better culture bacterial counts, physico-chemical and sensory characteristics compared to reconstituted commercial yogurt powder.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Song L. and Aryana K.J. 2014. Reconstituted yogurt from yogurt cultured milk powder mix has better overall characteristics than reconstituted yogurt from commercial yogurt powder. Journal of Dairy Science 97:6007-6015.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Vargas L. and Aryana K.J. 2014. Whey protein isolate improves acid tolerance and bile tolerances of Streptococcus thermophilus ST-M5 and Lactobacillus delbrueckii ssp. bulgaricus LB-12. Journal of Dairy Science (accepted).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: D. Olson, E. Gonzalez, M. Ponce, and K. J. Aryana, (2014). Effect of potassium sorbate and sodium benzoate concentrations on growth of cheese starter cultures. Abstract # M076. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 498 - 499.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: B. Mena and K. J. Aryana 2(2014). Influence of added lactose on some probiotic properties of yogurt culture bacteria. Abstract # M081. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 500-501.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: R. E. Brown and K. J. Aryana. (2014). Can prior subjection to pre-heating enhance the heat tolerance of mesophilic bacterial cultures? Abstract # 31. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 15.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: M. S�nchez-Vega and K. J. Aryana (2014). Influence of various health beneficial spices on some characteristics of yogurt culture bacteria and Lactobacillus acidophilus and sensory acceptability of spicy probiotic yogurt. Abstract # M079. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 499 - 500.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: M. Moncada*, C. Sabliov, C. Astete, and K. J. Aryana, (2014). Submicronization of sodium chloride and its effect on the physico-chemical and microbiological characteristics of surface salted cheese crackers. Abstract # M078. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 499.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: L. Song* and K. J. Aryana, (2014) Characteristics of yogurt manufactured using reconstituted yogurt cultured milk powder compared to yogurt powder. Abstract # M073. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 497.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: B. Mena and K. J. Aryana. (2014). Yogurt characteristics as effected by added lactose. Abstract # M080. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 500.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: M. Moncada*, C. Sabliov, C. Astete, and K. J. Aryana, (2014). Influence of submicronization of sodium chloride on the sensory characteristics of surface salted cheese crackers. Abstract # M077. ADSA annual meeting July 20-24, Kansas City, Missouri. J. Dairy Sci. Vol. 97, E-Suppl. 1, pp 499.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Foodindustry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has: 1) resulted in training 6 graduate students to date who either have graduated or will be graduating soon. 2) helped in training of several undergraduate students onthe dairy processing andlab analyses aspects. 3)helped developadditional interesting hypotheses. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest through 1) peer reviewed scientific publications and 2) presentation at national level scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Continueresearching on processing and ingredeints to enhance probiotic characteristics of dairy culture bacteria.

Impacts
What was accomplished under these goals? Consumers are becoming conscious of their diet, increasing protein intake and avoiding carbohydrates and fats. Whey proteins have branch chain amino acids responsible for muscle building. Whey protein isolate (WPI) contains more than 90% protein. The effect of incremental addition of WPI on probiotic characteristics of pure cultures and cultures in yogurt and yogurt characteristics are not known. The hypothesis was that “added” WPI will influence the characteristics of yogurt culture bacteria in pure form and in yogurt. The objectives were: to determine the influence of added WPI on (1) acid and bile tolerance, growth and protease activity of pure cultures Streptococcus thermophilus ST-M5 and Lactobacillus bulgaricus LB-12, (2) growth, acid and bile tolerance of starter culture from manufactured plain yogurt, (3) the physico-chemical characteristics of yogurt over its shelf life and (4) the sensory attributes of yogurt. WPI was used at 0, 1, 2 and 3% w/v. Acid tolerance was conducted on pure cultures and cultures from manufactured plain yogurt at 30 minutes intervals for 2 hours of incubation and bile tolerance at 1 hour intervals for 5 hours. Yogurt was manufactured using 0 (control), 1, 2 and 3% WPI. For sensory evaluation, blueberry yogurt was manufactured using the same WPI concentrations. Physico-chemical analyses of yogurts were conducted every 7 days during 35 days of storage. Enumeration of yogurt cultures during yogurt´s shelf life was evaluated at 7, 21 and 35 days of storage. Sensory evaluation was conducted on yogurt 7 days after its manufacture. Data were analyzed using Proc Mixed model of SAS® 9.3 program and by analysis of variance (ANOVA) using Proc GLM. Significant differences between means were analyzed at α = 0.05 using Tukey´s adjustment. Addition of whey protein isolate (WPI) had a positive effect on probiotic properties of yogurt starter bacteria and yogurt characteristics. Use of 1, 2 and 3% WPI resulted in significantly higher acid and bile tolerance for pure cultures Streptococcus thermophilus ST-M5 and Lactobacillus bulgaricus LB-12 for 120 minutes of exposure to acid and for 5 hours of exposure to bile. Use of 1, 2, and 3% WPI significantly increased growth of pure culture Streptococcus thermophilus ST-M5 at 24 hours of incubation compared to control. Use of 2 and 3% WPI resulted in significantly higher growth for pure Lactobacillus bulgaricus LB-12 from 24 to 60 hours of incubation compared to control and 1% WPI. Use of 1, 2 and 3% WPI significantly increased protease activity of both culture bacteria. Acid and bile tolerance of Streptococcus thermophilus ST-M5 and Lactobacillus bulgaricus LB-12 from manufactured fat free plain yogurt was evaluated at day 7 of storage. Use of 2% WPI significantly improved acid tolerance of Streptococcus thermophilus ST-M5 in yogurt. Bile tolerance of Lactobacillus bulgaricus LB-12 was significantly improved by use of 2 and 3% WPI compared to control over the 5 hours of bile exposure. Yogurts containing 2 and 3% WPI showed significantly higher pH and TA values compared to control. Apparent viscosity was not affected by WPI at 35 days of storage. Yogurts containing 1, 2 and 3% WPI resulted in significantly lower syneresis values (whey separation) compared to control over the entire storage period of 35 days. Addition of WPI had no effect on color and aroma of blueberry yogurt. Scores for taste, overall liking and acceptability were higher for 1 and 2% WPI. Addition of 1% WPI to yogurt contributed to higher scores for sensory thickness. Also the purchase intent increased with the addition of 1 and 2% WPI. Overall, 1 or 2% WPI can be recommended in manufacture of higher whey protein yogurts. Another study: Lactose sugar is a source of energy for Lactic Acid Bacteria (LAB) in dairy fermented products. Enrichment of yogurt with lactose addition may increase growth and viability of the yogurt starter culture (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) and enhance yogurt physico-chemical and sensory attributes. The objectives of this study were: to determine the influence of added lactose on (1) acid and bile tolerance of yogurt starter culture Streptococcus thermophilus ST-M5 and Lactobacillus bulgaricus LB-12, (2) the final lactose content of yogurt during its shelf life, (3) the physico-chemical characteristics of yogurt during shelf life, (4) the growth of the yogurt starter culture during yogurt´s shelf life and (5) the sensory attributes of yogurt. Acid tolerance test was conducted on pure culture at 30 minutes intervals for 2 hours of incubation period and bile tolerance at 1 hour intervals during 12 hours. Fat free plain set-type yogurt was manufactured using 0, 1, 3 and 5% w/w added lactose to accomplish objectives 2, 3 and 4. For objective 5, a blueberry yogurt was manufactured using the same lactose levels. Analyses for plain set-type yogurt were done at 7 days intervals during 35 days of storage period. Sensory evaluation was conducted on yogurt 3 days after its manufacture. Data were analyzed using Proc Mixed model of SAS® 9.3 program and by analysis of variance (ANOVA) using Proc GLM. Significant differences between means were analyzed at α = 0.05 using Tukey´s adjustment. Added lactose had a positive effect on probiotic properties of yogurt starter bacteria and yogurt characteristics. Added lactose at 3 and 5% w/w showed the highest acid tolerance for Streptococcus thermophilus ST-M5 and Lactobacillus bulgaricus LB-12 at 120 minutes of incubation. Added lactose at 5% w/w showed the highest bile tolerance for Lactobacillus bulgaricus LB-12 compared to the rest of treatments and for Streptococcus thermophilus ST-M5 added lactose at 1% w/w at 2 hours of incubation showed the highest bile tolerance. Growth of Streptococcus thermophilus ST-M5 significantly increased by lactose addition at 5% w/w in yogurt. Lactose addition did not have a significant effect on growth of Lactobacillus bulgaricus LB-12 in yogurt. Treatments containing 5% w/w added lactose showed the highest lactose content during storage period and had the lowest pH values. Treatment containing 5% added lactose showed the lowest viscosity values compared to the rest; and also the highest syneresis values over storage period of 35 days. Level of added lactose had no effect on appearance and color of blueberry yogurt. Scores for aroma were higher for control and 5% w/w added lactose. Samples containing added lactose showed higher scores for taste, sourness and sweetness. Lactose addition contributed to higher scores for overall linking. Also the acceptability of yogurts and purchase intent markedly increased with the addition of lactose.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Najim N and Aryana K.J. 2013. A mild pulsed electric field condition that improves acid tolerance, growth and protease activity of Lactobacillus acidophilus LA-K and Lactobacillus delbrueckii ssp. bulgaricus LB-12. Journal of Dairy Science 96: 3424-3434.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Abigail Greenbaum and K J. Aryana 2013. Effect of honey a natural sweetener with several medicinal properties on the attributes of a frozen dessert containing the probiotic Lactobacillus acidophilus. Open Journal of Medical Microbiology 3: 95-99.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xiang, B., Sundararajan, S., Mis Solval, K., Espinoza, L., Aryana, K., and S Sathivel. 2013. Effects of pulsed electric fields on physicochemical properties and microbial inactivation of carrot juice. J. Food Process. & Preserve. DOI: 10.1111/jfpp.12115
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: M. Sanchez-Vega and K. Aryana, (2013). Influence of some medicinal spices on the bile tolerance of Streptococcus thermophilus ST-M5. Abstract # T284. ADSA annual meeting July 8-12, Indianapolis, Indiana. J. Dairy Sci 96, E-Suppl 1, pp 99
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: M. Moncada, K. Aryana, C. E. Astete, and C. Sabliov. (2013). Production of sodium chloride nanoparticles by nanospray drying method. Abstract # T230. ADSA annual meeting July 8-12, Indianapolis, Indiana. J. Dairy Sci 96, E-Suppl 1, pp 81.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: B. Mena and K. Aryana, (2013) Influence of different concentrations of lactose on the growth of Lactobacillus delbrueckii ssp. bulgaricus LB-12 and Streptococcus thermophilus ST-M5. Abstract # W324. ADSA annual meeting July 8-12, Indianapolis, Indiana. J. Dairy Sci 96, E-Suppl 1 pp 333.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: L. Vargas and K. Aryana, (2013) Influence of whey protein isolate on growth of Streptococcus thermophilus ST-M5. Abstract # W325. ADSA annual meeting July 8-12, Indianapolis, Indiana. J. Dairy Sci 96, E-Suppl 1 pp 333.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: M. Sanchez-Vega and K Aryana, Effect of several health beneficial spices on the bile tolerance of Lactobacillus bulgaricus LB-12. (2013). Abstract # W326. ADSA annual meeting July 8-12, Indianapolis, Indiana. J. Dairy Sci 96, E-Suppl 1 pp 333.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: M. Rouse, C. A. Boeneke, K. V. OBrien*, and K. Aryana, (2013). Effect of oil-based and microencapsulated n-3 fatty acids on physical and chemical properties of processed cheese. Abstract #W225. ADSA annual meeting July 8-12, Indianapolis, Indiana. J. Dairy Sci 96, E-Suppl 1 pp 301.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Margie M. S�nchez-Vega, December 2013, INFLUENCE OF VARIOUS HEALTH BENEFICIAL SPICES ON SOME CHARACTERISTICS OF YOGURT CULTURE BACTERIA AND LACTOBACILLUS ACIDOPHILUS, AND SENSORY ACCEPTABILITY OF SPICY PROBIOTIC YOGURT. Ph.D. Dissertation. LSU.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Behannis J. Mena Chalas, August 2013. INFLUENCE OF ADDED LACTOSE ON PROBIOTIC PROPERTIES OF YOGURT CULTURE BACTERIA AND YOGURT CHARACTERISTICS. Masters thesis. LSU
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Luis Alfonso Vargas L�pez, December 2013. INFLUENCE OF ADDED WHEY PROTEIN ISOLATE ON PROBIOTIC PROPERTIES OF YOGURT CULTURE BACTERIA AND YOGURT CHARACTERISTICS. Masters thesis. LSU
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Lijie Song, December 2013. YOGURT CULTURED MILK POWDER AS A SUBSTITUTE FOR YOGURT POWDER. Masters thesis. LSU
  • Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: Maria Carolina Vives Habeych, May 2012. YOGURT CULTURES SURVIVE UPON EXPOSURE TO TWO ANTIMICROBIALS AND STREPTOCOCCUS THERMOPHILUS ST-M5 ACQUIRED RESISTANCE TO BOTH ANTIMICROBIALS. Masters thesis. LSU
  • Type: Theses/Dissertations Status: Published Year Published: 2011 Citation: Marvin Leonel Moncada Reyes, May, 2011., INFLUENCE OF LOW SONICATION INTENSITIES AT DIFFERENT TEMPERATURES ON ACID TOLERANCE, BILE TOLERANCE, PROTEASE ACTIVITY, AND GROWTH OF YOGURT CULTURE BACTERIA LACTOBACILLUS DELBRUECKII SSP. BULGARICUS AND STREPTOCOCCUS SALIVARIUS SSP. THERMOPHILUS. Masters thesis. LSU.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The information was shared with fellow dairy scientists through ten scientific publications. PARTICIPANTS: Aryana, K.J. (PI), Moncada M., Sanchez-Vega, M Mena, B Vives, M Boeneke, C Douglas Olson, Zhimin Xu, LSU AgCenter. TARGET AUDIENCES: Academia and the food and pharmaceutical industry. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Antimicrobials are added in the manufacture of some dairy products such as processed cheese. Commonly available Potassium Metabisulfite (PM) and Potassium Nitrite (PN) have been reported to have antimicrobial effect on pathogenic microorganisms such as Clostridium botulinum and Listeria monocytogenes. Yogurt is known for its health benefits, due to the presence of its culture bacteria. It is not known if yogurt culture bacteria can acquire resistance to antimicrobials. The objectives of this study were: 1. to elucidate the influence of PM and PN at various concentrations, on the growth of yogurt culture bacteria and 2. to determine the possible acquisition of resistance after prior exposure to low doses of these antimicrobials. For the first objective different concentrations of PM and PN were separately added to broth previously inoculated with Lactobacillus bulgaricus LB-12 and Streptococcus thermophilus ST-M5. Control samples did not receive any antimicrobial. For the second objective, treatments consisted of separately exposing cultures to 100 and 1,000ppm of both antimicrobials and after 24, 48 and 72 hours, transferring them into 10,000 and 100,000ppm, of both antimicrobials. Data were analyzed using Proc Mixed and Repeated Measures model of the Statistical Analysis System SAS. Differences of Least Square Means where used to determine significant differences. Neither PM nor PN had an antimicrobial effect on yogurt culture Lactobacillus bulgaricus LB-12 or Streptococcus thermophilus ST-M5. Prior exposure of Streptococcus thermophilus ST-M5 to both antimicrobials at 100 and 1,000ppm for 72 hours showed resistance to 10,000 and 100,000ppm of both PM and PN with a significant increase of 6 log CFU/mL compared to control. Consumers are on the look out for new dairy products and some dairy products have niche markets. Alcohol is used in manufacture of alcoholic eggnog. Appreciable quantity of alcohol (1 - 2.5%) is formed within dairy products kefir and koumiss because of the use of yeast cultures. Objectives were to study the effect of food grade ethanol on the growth of yogurt culture bacteria and the physico-chemical characteristics of yogurt. The ethanol was incorporated by stirring it into one day old plain yogurt. Product characteristics were studied weekly for a month of refrigerated (4C) storage. Data were analyzed using Proc Mixed model of Statistical Analysis System. The ethanol amount*storage period interaction effect was significant for Lactobacillus bulgaricus counts while the ethanol amount*storage period effect was not significant for Streptococcus thermophilus counts, viscosity, pH and titratable acidity (TA). Yogurts with ethanol, at these concentrations, can successfully be manufactured. Yogurt culture bacteria can survive in presence of alcohol as high as 7.5% v/v in yogurt and yogurt culture bacteria can develop resistance to some antimicrobials which opens up new possibilities for use of these health beneficial and culture bacteria in targeted applications.

Publications

  • Moncada M. and K. J. Aryana, 2012. Influence of Mild Sonication Conditions on the Characteristics of Streptococcus thermophilus ST-M5," Advances in Microbiology, 2: 8-16.
  • Moncada M and K. J. Aryana, 2012. Effect of mild sonication conditions on the attributes of Lactobacillus delbrueckii ssp. bulgaricus LB-12. Advances in Microbiology. 2: 104-111.
  • Rouse M,. Boeneke C. A., OBrien K and K. J. Aryana. 2012. Effect of oil based and microencapsulated omega-3 fatty acids on physical and chemical properties of processed cheese. Milk Science International. 67(3): 267-269.
  • Vives M and K. Aryana. 2012. Growth of yogurt culture bacteria in the presence of two antimicrobials. J. Dairy Sci. Vol. 95, E Suppl. 2 p 528.
  • Vives M., and K. Aryana. 2012. Acquired resistance of yogurt culture bacteria to two different antimicrobials. J. Dairy Sci. Vol. 95, E Suppl. 2 p 529.
  • Sanchez-Vega M., and K. Aryana 2012. Influence of various health beneficial spices on the acid tolerance of Streptococcus thermophilus ST-M5. J. Dairy Sci. Vol. 95, E Suppl. 2 p 529.
  • Moncada M.,and K. Aryana. 2012. Bile tolerance of Lactobacillus delbrueckii ssp. bulgaricus LB-12 subjected to mild sonication intensities at different temperatures. J. Dairy Sci. Vol. 95, E Suppl. 2 p 529-530.
  • Moncada M., and K. Aryana. 2012. Protease activity of Streptococcus thermophilus ST-M5 subjected to mild sonication intensities at different temperatures. J. Dairy Sci. Vol. 95, E Suppl. 2 p 530-531.
  • Mena B., and K. Aryana 2012. Influence of ethanol on some characteristics of stirred yogurt. J. Dairy Sci. Vol. 95, E Suppl. 2 p 278.
  • OBrien, K.V., C. A. Boeneke, K. J. Aryana, and W. Prinyawiwatkul. 2012. Viability of probiotic bacteria and yeasts in traditional and commercial kefir following frozen storage. J. Dairy Sci. Vol. 95, E Suppl. 2 p 651.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Masters thesis, 1 article in refereed journal, 7 presentations at national level scientific meetings, and undergraduate training were the research conducted on on this year's projects. PARTICIPANTS: K.J. Aryana (PI), D. Olson, K. Sternitzke, H. Paul, J. Boudreaux, and K. Moore, LSU AgCenter. TARGET AUDIENCES: Scientific community, dairy / food industry, PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
It is not known whether low homogenization pressures or mild sonication conditions can improve the probiotic characteristics of three important dairy cultures. In one study, the treatments were homogenization pressures of 0 MPa, 3.45 MPa (500 psi), 6.90 MPa (1000 psi), 10.34 MPa (1500 psi) and 13.80 MPa (2000 psi). Growth and bile tolerance of samples were determined hourly for 10 hours of incubation. Acid tolerance was determined every 20 minutes for 120 minutes of incubation. Protease activity was determined at 0, 12 and 24 hours of incubation. The experimental design was repeated measurements on complete randomized block. Data were analyzed using proc mixed model of statistical analysis system (SAS). Differences of least square means were used to determine significant differences at p<0.05 for main effect (homogenization pressure) and interaction effect (homogenization pressure * time). All low homogenization pressures improved acid tolerance of Lactobacillus delbrueckii ssp bulgaricus LB-12 but had no beneficial effect on protease activity and had negative effect on growth and bile tolerance of the bacterium. Low homogenization pressure of 6.90 MPa (1000 psi) improved the acid tolerance, bile tolerance, and protease activity; but homogenization pressures had no effect on growth of Streptococcus salivarius ssp thermophilus ST- M5. Low homogenization pressures of 13.80 MPa (2000 psi) and, 6.90 MPa (1000 psi) improved acid tolerance and bile tolerance, respectively, of Lactobacillus acidophilus LA-K but had no effect on protease activity and growth of the bacterium. Some low homogenization pressures positively influenced some characteristics of yogurt culture bacteria and Lactobacillus acidophilus LA-K. In another study, the treatments were four sonication intensities of 8.07, 14.68, 19.83 and 23.55 Watts/cm2 randomized at three different temperatures (4, 22 and 40C) of inoculated peptone water before sonication. Low sonication conditions include a) low sonication intensities, b) temperatures and c) times; all three of which played a role in influencing the desirable attributes of both microorganisms. Of all the low sonication intensities studied, 14.68 watts /cm2 had the best overall influence at certain time points for Streptococcus thermophilus, improving its acid tolerance, bile tolerance and growth at 4C, growth at 22C, bile tolerance and growth at 40C, and improving the Lactobacillus bulgaricus bile tolerance and growth at 4C, its acid tolerance and protease activity at 40C. Low sonication intensity of 19.83 Watts/cm2 had the overall best influence at certain time points for acid tolerance of both microorganisms at 22C. Low sonication intensity of 23.55 Watts/cm2 had the overall best influence at certain time points for protease activity of Streptococcus thermophilus at 40C and Lactobacillus bulgaricus at 22C. Some low sonication conditions improved certain characteristics of culture bacteria. Some low homogenization pressures and some low sonication conditions improved certain probiotic characteristics of dairy culture bacteria hence can be used to pre treat dairy cultures to improve their desirable / probiotic characteristics.

Publications

  • T. Muramalla and K.J. Aryana. 2011. Some low homogenization pressures improve certain probiotic characteristics of yogurt culture bacteria and Lactobacillus acidophilus LA-K. Journal of Dairy Science. 94: 3725-3728.
  • C. Boeneke, J. Vargas and K. Aryana 2011. Fluid milk quality survey. Abstract # T70. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 290.
  • M. Moncada and K. Aryana. 2011. Effect of low sonication intensities on the growth of Streptococcus thermophilus ST-M5 subjected to different temperatures. Abstract # T83. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 293.
  • M. Moncada and K. Aryana. 2011. Low sonication intensity influences on the protease activity of Lactobacillus delbrueckii ssp. bulgaricus LB-12 at different temperatures. Abstract # T84. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 293-294.
  • M. Moncada and K. Aryana. 2011. Influence of low sonication intensities at different temperatures on the bile tolerance of Streptococcus thermophilus ST-M5. Abstract # T85. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 294.
  • N. Najim and K. Aryana. 2011. Screening of pulsed electric field parameters for enhancing acid tolerance of Streptococcus thermophilus ST-M5. Abstract # T86. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 294.
  • N. Najim and K. Aryana. 2011. Mild pulsed electric field conditions identified for improving growth, protease activity and acid tolerance of Lactobacillus delbrueckii ssp. bulgaricus LB-12 and Lactobacillus acidophilus LA-K. Abstract # T87. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 294-295.
  • N. Najim and K. Aryana. 2011. Impact of mild pulsed electric field conditions on improving bile tolerance, protease activity and growth of Streptococcus thermophilus ST-M5. Abstract # T88. Journal of Dairy Science, Vol 94. E Supplement 1. pp. 295.