Mitigation of Undesirable Flavor in Kefir Intended for Adjuvant Treatment of Clostridioides difficile Infection
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University
By
Megan Kathleen Kesler
Graduate Program in Food Science and Technology
The Ohio State University
2019
Master's Examination Committee:
Valente B. Alvarez, Advisor
Rafael Jiménez-Flores
Ahmed Yousef
Copyrighted by
Megan Kathleen Kesler
2019
Abstract
Clostridioides difficle infection (CDI) is the most common health-care associated infection in the United States. Annually, CDI infects nearly half a million patients, kills
29,000 and costs the healthcare system an estimated US$6.3 billion. Although this pathogen can be treated with antibiotics, in 25% of cases, the bacterium does not respond to antibiotics and the patient develops a recurrent infection. Due to the hardy nature of the bacteria causing the recurrent CDI, additional, more costly treatment methods such as fecal microbiota transplantation must be employed. Alternatively, consumption of kefir, a commercially available fermented dairy beverage with live probiotic cultures, was recently found to be effective in curing recurrent CDI when consumed alongside the prescribed antibiotic treatment. Unfortunately, kefir has limited patient acceptance due to its strong acidic and fruity flavors resulting from the fermentation process. To combat this, kefir may be further processed using freeze-drying or vacuum evaporation to remove undesirable volatile organic compounds (VOCs) that greatly contribute to flavor, but heat during processing must be minimized to avoid reducing the population of beneficial microorganisms. In this study, effect of vacuum evaporation and freeze-drying on kefir
VOC concentration, microbial viability and activity, and sensory quality was assessed.
Commercially manufactured kefir was subjected to either a vacuum evaporation or freeze-drying treatment, or was not further processed (control). Loss of volatile compounds was monitored at the ppb level using selected-ion flow-tube mass ii spectrometry (SIFT-MS); kefir acceptability was evaluated by untrained panelists using a
9-point hedonic scale, and differences among treatments was determined by comparing mean scores; microbial viability was assessed using selective media for enumeration of
Lactobacillus spp. and Lactococcus spp.; and microbial activity was assessed by measuring fermentation rate of milk inoculated with treated and untreated (control) kefir.
Vacuum evaporation and lyophilization treatments significantly diminished VOC content in the kefir for 26 out of 27 compounds (P < 0.05). Cumulative VOC concentration for all 27 compounds was reduced by approximately 62% after both vacuum evaporation and freeze-drying treatments. Despite the considerable difference in kefir VOC content, vacuum evaporation and lyophilization treatments did not significantly improve the liking of commercial kefir in sensory tests. The concentrations of Lactobacillus spp. and
Lactococcus spp. present in the kefir were significantly reduced by freeze-drying and vacuum evaporation treatments. Although significant, the population of lactic acid bacteria (LAB) in the kefir samples was still considered sufficient for probiotic products.
Fermentation rates were slightly reduced after the experimental treatments, with lyophilization having a more significant effect.
iii
Acknowledgments
It is with immense gratitude that I acknowledge the support and help of my advisor, Dr. Valente Alvarez. His guidance and encouragement throughout the project were vital to my success. It was an honor and a pleasure to be mentored by him. I would also like to thank Dr. Rafael Jiménez-Flores for encouraging me to think like a scientist and for treating me as an extended part of his workgroup, Dr. Ahmed Yousef for his time and helpful suggestions, and Dr. Cory Hussain for bringing this project to our group and for his continuing support. I am indebted to Dr. Hardy Castada for his time and assistance, and for his help in developing my methodology. Thank you also to Dr. Israel
García-Cano, Dr. Diana Rocha-Mendoza, and Dr. Joana Ortega-Anaya for the many times they helped me in the laboratory.
My deepest gratitude to Heather Bell, Steven Simmons, Gary Wenneker, Matt
Papic, Stelios Sarantis and Tori Dong for their assistance, moral support, and friendship.
My research would not have been possible without their help, and my time in the department would not have been as bright without their company. I will miss working with them.
Thank you to all of my friends, old and new, for your encouragement and for the fun times that lifted my spirits when I needed it most. I would especially like to thank
Elliot Dhuey for helping me navigate graduate school every step of the way.
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A special thank you to my husband, Ryan, for his unwavering love and support, and for all of the sacrifices he made so that I could achieve this degree. He tirelessly cheered me on and never let me give up. I could not have done it without him by my side.
Last but not least, I would like to thank my family. It is because of them that I am where I am today.
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Vita
June 2012 ...... Minnetonka High School
May 2017 ...... B.S. Food Science and Technology,
University of Wisconsin - Madison
August 2017 to present ...... Graduate Research Associate, Department
of Food Science and Technology, The Ohio
State University
Fields of Study
Major Field: Food Science and Technology
vi
Table of Contents
Abstract ...... ii
Acknowledgments...... iv
Vita ...... vi
List of Tables ...... x
List of Figures ...... xi
Chapter 1: Introduction ...... 1
Chapter 2: Literature Review ...... 4
2.1 Kefir ...... 4
2.1.1 Introduction to Fermented Milk Products ...... 4
2.1.2 Origin and History ...... 5
2.1.3 Definition and Composition ...... 6
2.1.4 Manufacture ...... 9
2.1.5 Microflora ...... 11
2.1.6 Sensory Characteristics...... 13
2.1.7 Therapeutic Properties ...... 17
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2.2 Clostridioides difficile Infection...... 19
2.2.1 Background ...... 19
2.2.2 Treatment Methods ...... 21
2.2.3 Kefir and Clostridioides difficile Infection ...... 23
2.3 Kefir Post-Manufacture Processing ...... 24
2.3.1 Application of Vacuum Drying Technologies for Removal of VOCs ...... 25
2.3.2 Lyophilization ...... 25
2.3.3 Vacuum Evaporation ...... 28
2.4 Methods of Analyses: Selected-Ion Flow-Tube Mass Spectrometry ...... 29
Chapter 3: Materials and Methods ...... 31
3.1 Stock Solution Preparation ...... 31
3.2 Sample Preparation ...... 31
3.2.1 Control Samples ...... 31
3.2.2 Vacuum Evaporation ...... 31
3.2.3 Lyophilization ...... 32
3.3 Methods of Analysis...... 34
3.3.1 Moisture Analysis ...... 34
3.3.2 Selected-Ion Flow-Tube Mass Spectrometry ...... 34
3.3.2.1 Identification of Potential Kefir Compounds ...... 34
viii
3.3.2.2 Sample Preparation and SIFT-MS Analysis ...... 37
3.3.3 Sensory Evaluation ...... 38
3.3.4 Enumeration of Viable Microorganisms ...... 39
3.3.5 Fermentation Rate...... 40
3.3.6 Statistical Analysis ...... 40
Chapter 4: Results and Discussion ...... 41
4.1 Selected-Ion Flow-Tube Mass Spectrometry ...... 41
4.2 Sensory Evaluation ...... 54
4.3 Enumeration of Viable Microorganisms ...... 57
4.4 Fermentation Rate...... 62
Chapter 5: Conclusion...... 65
References ...... 68
Appendix : Additional Selected-Ion Flow-Tube Mass Spectrometry Results ...... 81
ix
List of Tables
Table 1. Chemical composition of kefir (Arslan 2015)...... 7
Table 2. Volatile fermentation products detected in kefir by GC-MS (Walsh et al. 2016).
...... 15
Table 3 Therapeutic properties claimed for kefir (Frias et al. 2017)...... 18
Table 4. CDI Treatment Strategies. Abbreviations: d, day; hr, hours, PO, per os (oral); q, every; qd, daily; qod, every other day. (Cole and Stahl 2015)...... 22
Table 5. Kinetics of the volatile compounds measured using the selected ion mode (SIM) scan method of the SIFT-MS...... 35
Table 6. Volatile compounds measured using the selected ion mode (SIM) scan method of the SIFT-MS and their odor descriptors and boiling points at atmospheric pressure
(Walsh et al. 2016; Kim et al. 2019)...... 42
x
List of Figures
Figure 1. Macroscopic structure of fresh kefir grains (Leite et al. 2013)...... 7
Figure 2. Structure of kefiran (Prado et al. 2015)...... 8
Figure 3. Flow diagram for kefir production (Frias et al. 2017)...... 10
Figure 4. Effect of storage at 5 h () and 15 h () on kefir attributes (Wszolek et al.
2001)...... 17
Figure 5. Phase diagram of water (Knudsen 2011)...... 26
Figure 6. State diagram of sucrose solution demonstrating the onset of the glass transition, Tg, in maximally freeze-concentrated solution (Roos 2010)...... 28
Figure 7. Randomized placement of 9 kefir samples in lyophilizer. A: Stock A, B: Stock
B, C: Stock C...... 33
Figure 8. Concentration of acetaldehyde in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 46
Figure 9. Concentration of acetone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15). Means with different letters are significantly different (P < 0.05)...... 47
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Figure 10. Concentration of butanoic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 48
Figure 11. Concentration of dimethyl sulfide in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 49
Figure 12. Concentration of ethyl acetate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 50
Figure 13. Concentration of formic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 52
Figure 14. Concentration of propanal in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 53
Figure 15. Spider plot of sensory attribute hedonic scores for kefir samples (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely). *P < 0.05...... 55
Figure 16. Histogram distribution of overall acceptability scores for kefir samples (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely)...... 56
Figure 17. Population of Lactobacillus spp. in kefir samples. Data are means and standard errors from three separate trials, with two samples analyzed per treatment (n = 6). Means with different letters are significantly different (P < 0.05)...... 59
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Figure 18. Population of Lactococcus spp. in kefir samples. Data are means and standard errors from three separate trials, with two samples analyzed per treatment (n = 6). Means with different letters are significantly different (P < 0.05)...... 60
Figure 19. Fermentation rate of UHT milk inoculated with 4% (v/v) experimental kefir
(untreated (control), vacuum evaporated or lyophilized), incubated at 29°C for 12 hours.
Data are means and standard errors from three separate trails (n=3). Asterisks indicate values that are significantly different (P < 0.05) from corresponding values for untreated samples (control)...... 63
Figure 20. Concentration of 2-methylbutanoic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 81
Figure 21. Concentration of 2-pentanone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 82
Figure 22. Concentration of 2-phenylethanol in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 83
Figure 23. Concentration of dimethylsulfone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 84
xiii
Figure 24. Concentration of ethyl butanoate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 85
Figure 25. Concentration of ethyl hexanoate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 86
Figure 26. Concentration of isoamyl acetate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 87
Figure 27. Concentration of pyrazine in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 88
Figure 28. Concentration of 2,3-butanedione in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 89
Figure 29. Concentration of 2-methyl-1-butanol in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 90
Figure 30. Concentration of 2-methylfuran in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 91
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Figure 31. Concentration of butanone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 92
Figure 32. Concentration of carbon disulfide in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05)...... 93
Figure 33. Concentration of methanol in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 94
Figure 34. Concentration of methional in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P ...... 95
Figure 35. Concentration of pentanal in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15). Means with different letters are significantly different (P < 0.05)...... 96
Figure 36. Concentration of pentanoic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 97
Figure 37. Concentration of acetic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05)...... 98
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Figure 38. Concentration of benzene in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15). Means with different letters are significantly different (P < 0.05)...... 99
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Chapter 1: Introduction
According to the Centers for Disease Control and Prevention, nearly half a million people contract Clostridioides difficile Infection (CDI) each year in the United
States, and annual CDI related expenses total an estimated US$6.3 billion (Zhang et al.
2016). Of those infected, 29,000 die within 30 days and about half of the deaths are directly attributable to CDI. Most commonly, hospitalized patients contract CDI when they are on antibiotics for another ailment (Caroff et al. 2017). Subsequently, an additional oral antibiotic is prescribed to treat the CDI (Teasley et al. 1983; Bricker et al.
2005). However, in 20-30% of cases the treatment is not effective and the patient develops a recurrent infection (Gerding et al. 2008; Cole and Stahl 2015). Due to the difficulty of eradicating Clostridioides difficile bacteria with conventional means, additional, more costly treatment methods, such as fecal microbiota transplantation, must be employed (van Beurden et al. 2017).
Alternatively, consumption of kefir, a commercially available dairy beverage with live probiotic cultures, was recently found to be effective in curing recurrent CDI when consumed alongside the prescribed antibiotic treatment (Bakken 2014). Kefir originates from the Caucasus Mountain region in Eastern Europe and is fermented using kefir grains, which are small white granules composed of polysaccharide, caseins, and symbiotic colonies of bacteria and yeast (Stepaniak and Fetliński 2002; Guzel-Seydim et al. 2005). Kefir as a whole, along with many of the microorganisms isolated from kefir,
1 has been associated with numerous health benefits including immunomodulatory effects
(Osada et al. 1993; Vinderola et al. 2005), antitumoral, antimutagenic, and anticarcinogenic activities (De Moreno De Leblanc et al.; Liu et al. 2002), anti-diabetic effects (Kwon et al. 2006; Punaro et al. 2014), anti-inflammatory effects (Rodrigues et al.
2005b; Lee et al. 2007), antimicrobial properties (Santos et al. 2003; Silva et al. 2009), healing (Rodrigues et al. 2005a; Huseini et al. 2012), and cholesterol-lowering effects
(Liu et al. 2006; Wang et al. 2009).
Unfortunately, kefir has limited acceptance among Western consumers due to its strong acidic and unusual flavors resulting from the fermentation process. Volatile organic compounds (VOCs) are a group of low molecular weight aliphatic and aromatic compounds that have a high vapor pressure at room temperature (Fleming-Jones and
Smith 2003). VOCs are responsible for aroma in foods, and aroma is an important factor that contributes to overall consumer acceptance of a food or beverage. There is very little information in the literature regarding which compounds are responsible for undesirable flavors and aromas in kefir. However, to combat undesirable flavors in kefir, aroma chemicals as a whole, both acceptable and objectionable, could be removed from the product to create a bland product that may then be flavored to fit consumer preference.
To separate undesirable VOCs, kefir may be further processed using freeze-drying and vacuum evaporation, vacuum drying technologies commonly employed by the food industry.
The overall goal of this study is to determine whether freeze-drying and vacuum evaporation can improve the acceptability of kefir without affecting the probiotic quality
2 of the product. The specific objectives of this study are 1) To evaluate the efficiency of freeze-drying and vacuum evaporation processes to remove VOCs from kefir, 2) To investigate the impact of VOCs on the sensory quality of kefir, and 3) To determine the effect of freeze-drying and evaporation processes on the probiotic content of kefir. The hypotheses corresponding to these objectives are 1) Vacuum evaporation and freeze- drying will significantly separate VOCs in kefir due to the low pressures used in the processes; vacuum evaporation will separate VOCs more effectively than freeze-drying due to the higher temperatures used in this process, 2) Removal of VOCs from kefir will improve acceptability of kefir because VOCs play an important role in food aroma and flavor, and 3) Freeze-drying will significantly reduce the concentration of viable microorganisms present in kefir because of low-water stress caused by freezing and drying, and intracellular ice formation; and that vacuum evaporation would not considerably diminish the population of microorganisms in kefir because the operating temperature is not lethal for kefir cultures.
3
Chapter 2: Literature Review
2.1 Kefir
2.1.1 Introduction to Fermented Milk Products
Cultured dairy products are milk products that have been fermented by lactic acid bacteria (Aryana and Olson 2017). Under microaerophilic conditions and small degrees of heat, the lactic acid bacteria convert lactose in the milk into lactic acid. The production of acid lowers the milk’s pH to or below 4.6, the isoelectric point of casein, causing coagulation of casein micelles and gel formation (Lucey and Singh 2003). The low pH of fermented dairy products also inhibits the growth of pathogenic bacteria and spoilage microorganisms, rendering the milk safe and increasing the product shelf life. The safe nature of fermented milk made consumption of cultured dairy products attractive to primitive cultures throughout the world (Aryana and Olson 2017). Thus, various cultured dairy products developed in virtually all regions where humans consumed mammalian milk. Product characteristics differed because of various factors, including mammalian species that produced the milk, microorganisms naturally present in the environment that served as the starter cultures, additional ingredients added to the milk, and manufacturing processes utilized.
Many popular cultured dairy products, including yogurt, cultured buttermilk, and sour cream, are fermented by lactic acid bacteria alone. However, kefir, koumiss, and laban are fermented by a microbial cocktail that includes lactic acid bacteria, other 4 bacteria such as acetic acid bacteria, and yeast (Fleet 1990; Muir et al. 1999). The presence of yeast and acetic acid bacteria in the starter culture results in product characteristics, including flavor, odor, and consistency, which are distinct from those found in dairy products fermented only with lactic acid bacteria. In addition, various putative health benefits have been linked to consumption of dairy products fermented by a diverse microbial cocktail. Due to its unique sensory properties and purported health benefits, kefir in particular, has grown in popularity throughout the world in recent years
(Leite et al. 2013; Nalbantoglu et al. 2014; John and Deeseenthum 2015).
2.1.2 Origin and History
Kefir originates from the Caucasus Mountain region in Eastern Europe, where families have been making it for centuries (Stepaniak and Fetliński 2002). Traditionally, kefir was made at home by individual families as goat or cow’s milk was fermented in leather sacks, clay pots, or oak barrels (Özer and Özer 1999; Stepaniak and Fetliński
2002). The fermentation vessel, which was kept inside during the winter and outside in the summer, was continually fermented and supplied with fresh milk whenever fermented product was removed. In time, an insoluble, spongy layer would form along the lining of the sack or barrel. This layer could be removed from the sack, broken up, and dried to form kefir grains that could maintain their activity for years (Leite et al. 2013; Frias et al.
2017). These grains became known as the “Prophet’s millet” because some say the
Prophet Mohammed gave the grains, a symbol of eternal life, to a select tribe (Stepaniak and Fetliński 2002). Industrial manufacture of kefir began at the end of the nineteenth century in Russia and countries of the former Soviet Union, where it grew in popularity,
5 eventually becoming a household staple (Frias et al. 2017). Since then, commercial manufacture of kefir has spread to many other countries in Europe, Asia, and North
America.
2.1.3 Definition and Composition
The name kefir, which is also known as kefyr, kephir, kefer, kiaphur, kepi, or kippi, is derived from the Turkish word for inebriating or fermenting - ‘ker’ (Stepaniak and Fetliński 2002; Leite et al. 2013). Kefir is a dairy beverage that is fermented using kefir grains, which are small, water insoluble, irregular white/yellow granules approximately 0.2-2 cm in size, consisting of polysaccharide, caseins, and symbiotic colonies of bacteria and yeast (Stepaniak and Fetliński 2002; Guzel-Seydim et al. 2005).
As shown in Figure 1, attributed to Leite et al. (2013),the grains resemble cauliflower florets.
The grains contain 85-90% water and approximately 57% carbohydrate, 33% protein, 4% fat, and 6% ash (Stepaniak and Fetliński 2002). However, composition of kefir differs due to type of milk used and production methods. The average chemical composition of kefir is shown in Table 1, attributed to Arslan (2015).
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Figure 1. Macroscopic structure of fresh kefir grains (Leite et al. 2013).
Component Average (%)
Moisture 89.0-90.0
Carbohydrate 6.0
Protein 3.0
Lipid 0.2
Ash 0.7
Table 1. Chemical composition of kefir (Arslan 2015).
The polysaccharide, called kefiran, is a water-soluble glucogalactan that serves as a protective matrix for the microorganisms, which excrete the substance as they
7 reproduce (Piermaria et al. 2008). Figure 2, attributed to Prado et al. (2015) shows the chemical structure of kefiran.
Figure 2. Structure of kefiran (Prado et al. 2015).
The Codex Alimentarius defines kefir as a fermented milk and specifies that the starter culture for kefir is to be prepared from kefir grains, Lactobacillus kefiri, species of the genera Leuconostoc, Lactococcus and Acetobacter growing in a strong specific relationship. The sum of microorganisms constituting the starter culture must be a minimum of 107 cfu/g, in total. Furthermore, it states that kefir grains constitute both lactose fermenting yeast (Kluyveromyces marxianus) and non-lactose-fermenting yeasts
(Saccharomyces unisporus, Saccharomyces cerevisiae and Saccharomyces exiguus). The concentration of yeast must be a minimum of 104 CFU/g. (Codex Alimentarius
8
Commission 2003). The Codex Alimentarius also states that kefir must be composed of a minimum of 2.7% (m/m) milk protein, less than 10% (m/m) milk fat, and a minimum of
0.6% (m/m) titratable acidity expressed as % lactic acid. No specification is given for ethanol content (Codex Alimentarius Commission 2003). Traditionally manufactured kefir made with kefir grains may contain up to 2% ethanol, but industrially produced kefir only contains 0.01-0.1% ethanol (Stepaniak and Fetliński 2002).
2.1.4 Manufacture
Kefir can be made from kefir grains (traditional method), freeze-dried kefir starter culture (industrial method), or by backslopping from finished product (Russian/European method) (Bensmira et al. 2010). Figure 3, attributed to Frias et al. (2017) shows the process for each method of kefir production. Different kinds of milk (i.e. cow, goat, sheep) can be used to make kefir, however, full-fat, low-fat, or non-fat cow’s milk is typically used for industrial manufacture (Wszolek et al. 2001; Frias et al. 2017).
Milk used for kefir is heated to denature whey proteins, which increases their water holding capacity. Thus, the finished product will have less syneresis and improved consistency. Heat treatment of milk also serves to inactivate lipase, reduce redox potential, and increase the availability of nutrients for the growth of the kefir starter culture (Sarkar 2008; Frias et al. 2017). In traditional manufacture kefir grains are added to the heat-treated milk at a concentration of 2-10%, and then the milk is fermented at 20-
25°C for 18-24 hours. After fermentation, the grains are sieved out to be reused. Finally, the finished product is cooled and packaged. In the Russian method, milk is inoculated
9 from a “mother culture,” which is simply milk fermented by grains with the grains removed (Farnworth 2003).
Figure 3. Flow diagram for kefir production (Frias et al. 2017).
The steps for industrial manufacture of kefir are similar to traditional manufacture apart from a few key differences. First, the milk is standardized and homogenized before heat treatment. Next, rather than inoculating milk with fresh kefir grains, a freeze-dried
10 starter culture is used. The freeze-dried starter may either be lyophilized kefir grains, or a cocktail of pure cultures isolated from the grains in combination with commercially available cultures (Beshkova et al. 2002; Leite et al. 2013). Finally, a maturation step after fermentation encourages growth of yeast that contribute to flavor and allows whey proteins to absorb more water (Frias et al. 2017).
2.1.5 Microflora
Kefir grains are symbiotic microbial communities of complex nature, arising from the physical association of approximately fifty species of bacteria and yeast (Stepaniak and Fetliński 2002; Frias et al. 2017). The microbial composition of kefir grains varies between batches of kefir due to differences in media used, grain to milk ratio, cultures used, incubation time and temperature, extent of agitation, and type of package and storage conditions (Sarkar 2008). However, the ratio of yeasts to lactic acid bacteria is relatively stable. Furthermore, the ratios of heterofermentative to homofermentative and mesophilic to thermophilic species are also generally consistent between grains
(Stepaniak and Fetliński 2002). Lactic acid bacteria are the principal microorganism present in kefir, accounting for 65-80% of the microorganisms in the grain (Witthuhn et al. 2005). Some lactic acid bacteria that have been found in kefir include Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus kefiranofaciens, Lactobacillus kefirgranum, Lactobacillus casei, Lactobacillus paracasei,
Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus parakefiri, Lactococcus lactis, Leuconostoc mesenteroides,
Leuconostoc gelidum, and Leuconostoc kimchi (Witthuhn et al. 2005; Walsh et al. 2016;
11
Frias et al. 2017). Yeasts isolated from kefir grains include Kluyveromyces marxianus,
Kluyveromyces lactis, Debaryomyces hansenii, Debaryomyces occidentalis, Torulaspora delbrueckii, Pichia fermentans, Torula kefir, Kazachstania unispora, Kazachstania exigua, Saccharomyces exiguous, Saccharomyces cerevisiae, Saccharomyces turicensis,
Issatchenkia orientalis, Candida kefir and Candida lambica (Witthuhn et al. 2005; Leite et al. 2013; Frias et al. 2017). Acetic acid bacteria have been isolated only from some kefirs. However, this may be because cultivation and isolation of acetic acid bacteria from foods is difficult. Acetic acid bacteria are often present, but their cells are not in a culturable state (De Roos and De Vuyst 2018). Species of acetic acid bacteria that have been isolated from kefir include Acetobacter pasteurianus, Acetobacter aceti, and
Acetobacter rasens (Witthuhn et al. 2005). Geotrichum candidum – a mycelial fungus, has also been isolated from kefir grains (Pintado et al. 1996). It is likely that many species of lactic acid bacteria, acetic acid bacteria, and yeast present in kefir remain unidentified.
The matrix of microorganisms in kefir grains is non-homogeneous. For example,
Lactobacillus kefir is primarily found in outer layers of the grain, while Lactobacillus kefiranofaciens tends to reside in the center of the grain. Yeasts are mainly located at the core of the grain, although lactose-fermenting yeasts exist in the peripheral layers
(Stepaniak and Fetliński 2002; Frias et al. 2017)
The microflora of the fermented kefir product differs from kefir grains. When kefir grains are incubated in milk, the kefir grains grow and some of the microorganisms from the grains are shed into the milk, where they continue to grow individually.
12
However, just as is the case in the grains, the microorganisms in kefir are dependent on each other for their growth and survival while fermenting the milk as the produced metabolites can stimulate or inhibit the growth of other microorganisms (Lopitz-Otsoa et al. 2006). Production of lactic acid by lactose fermenting bacteria, for example, creates an environment suitable for yeast growth. Additionally, amino acids and fatty acids produced by some proteolytic and lipolytic yeasts, and vitamin B produced by
Acetobacter spp. provides nutrients for microbial growth (Rea et al. 1996; Lopitz-Otsoa et al. 2006). A study that examined changes in microbial composition of kefir over the course of 24-hour fermentations using three kefir grains from separate geographic locations reported that kefir fermentation occurs in succession (Walsh et al. 2016). The bacterial communities of the three kefirs all followed the same pattern of succession: during the first 8 hours of fermentation, Lactobacillus, Leuconostoc, and Acetobacter populations all increased. At 8 hours, Lactobacillus kefiranofaciens dominated; between
8 and 24 hours, L. kefiranofaciens decreased, while Leuconostoc mesenteroides,
Leuconostoc citreum, Leuconostoc gelidum, Leuconostoc kimchi, Lactobacillus helveticus, and Acetobacter pasteurianus increased. Fungal succession patterns were not as clear, as they varied between kefir samples. At the end of the fermentation, 98% of the bacterial population was either Lactobacillus spp., Leuconostoc spp. or Acetobacter spp, while 99% of the fungal population was Saccharomyces spp. or Kazachstania spp.
2.1.6 Sensory Characteristics
Kefir fermentation results in the production of metabolites that alter milk and give kefir its unique chemical, physical, and sensory properties. The major metabolite of kefir
13 fermentation is lactic acid produced by the lactic acid bacteria. Thus, kefir is characterized as being highly acidic and sour. Homofermentative lactic acid bacteria produce lactic acid; heterofermentative lactic acid bacteria produce lactic acid and carbon dioxide. Lactic acid lowers the pH to or below 4.6 causing gelation of casein micelles and giving kefir a smooth, viscous consistency. Ethanol and carbon dioxide produced by the yeast make kefir a slightly alcoholic, self-carbonated beverage. The yeast also impart a distinct fermented flavor to kefir. Additional fermentation products in kefir include acids, alcohols, aldehydes, esters, ketones, and sulfur compounds. The presence of each of these compounds in kefir contributes to the complex flavor and aroma of the product. Specific odor descriptors for compounds found in kefir are listed in Table 2, attributed to Walsh et al. (2016).
Commercially manufactured kefir contains approximately 0.04-0.15% acetic acid,
≤ 0.4% CO2, 1-4mg/L diacetyl and several milligrams/L of acetaldehyde (Stepaniak and
Fetliński 2002). It has been reported that the optimum flavor balance occurs when the ratio of diacetyl to acetaldehyde is 3:1 (Muir et al. 1999). Defects in fermentation that alter kefir flavor and aroma may occur. One defect common in commercially produced kefirs is an unpleasant “yeasty” flavor and aroma, caused by rapid growth of
Saccharomyces cerevisiae. Another defect is a strong vinegar aroma caused by excessive production of acetic acid. Additionally, a bitter taste may result if Geotrichum candidum or some atypical yeasts are present (Leite et al. 2013).
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Table 2. Volatile fermentation products detected in kefir by GC-MS (Walsh et al. 2016).
To date, very limited information exists in the literature about sensory characteristics of kefir. Muir et al. (1999) compared the sensory properties of different types of Polish kefir and buttermilk with plain yogurt and buttermilk of UK origin. Two types of Polish kefir were used for the study: “traditional kefir” was made using kefir grains, and was associated with the production of alcohol and large amounts of CO2,
15 while “modified kefir” was made from a blend of microorganisms consisting of lactococci, lactobacilli and yeast, and was very lightly carbonated. They found that traditional kefir was similar to the buttermilk, which were both perceived as acid/sour and bitter by trained panelists. The modern kefir had a lower acid/sour rating, and a higher creamy flavor rating. Additionally, the researchers found that panelists were positively influenced by creamy and viscous attributes, and were negatively influenced by acid/sour, bitterness, and serum separation. Walsh et al. (2016) conducted sensory acceptance evaluation and ranking descriptive analysis for kefirs made from three kefir grains originating from France, Ireland, and the United Kingdom and found that samples with a likeable flavor were associated with buttery notes, whereas samples with a less likeable flavor were associated with fruity notes. Wszolek et al. (2001) studied differences in sensory attributes of kefirs made from bovine, caprine or ovine milk and reported that milk species had a significant effect on sensory character of the kefir.
Overall, panelists preferred the flavor of kefirs made with bovine and ovine milk to those made with caprine milk. The authors also found that type of starter culture used (either traditional grains preserved in saline solution or two different freeze-dried cultures) had an effect on overall acceptability and flavor. The effect of storage on intensity of sensory attributes was also examined (Figure 4, attributed to Wszolek et al. (2001)).
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Figure 4. Effect of storage at 5 h () and 15 h () on kefir attributes (Wszolek et al.
2001).
2.1.7 Therapeutic Properties
Historically, kefir has been associated with numerous health benefits and medicinal properties (Table 3). Modern research has verified and explained some of these claims, but not all. Additionally, the numerous origins and manufacturing conditions of kefirs that have been studied make corroboration of reported results difficult. Despite this, some of the main therapeutic benefits that have been linked with kefir consumption
17 are immunomodulatory effects (Osada et al. 1993; Vinderola et al. 2005), antitumoral, antimutagenic, and anticarcinogenic activities (De Moreno De Leblanc et al.; Liu et al.
2002), anti-diabetic effects (Kwon et al. 2006; Punaro et al. 2014), anti-inflammatory effects (Rodrigues et al. 2005b; Lee et al. 2007), antimicrobial properties (Santos et al.
2003; Silva et al. 2009), healing (Rodrigues et al. 2005a; Huseini et al. 2012), and cholesterol-lowering effects (Liu et al. 2006; Wang et al. 2009).
Table 3 Therapeutic properties claimed for kefir (Frias et al. 2017).
Kefir has a high nutritional value due to its chemical and nutrient composition.
Like milk, kefir is a good source of calcium and protein (Farnworth 2003). In addition, the fermentation process improves the quantity and bioavailability of nutrients present in the milk. For example, proteolytic enzymes hydrolyze milk proteins during kefir fermentation releasing free amino acids. It has been reported that kefir has higher
18 amounts of threonine, serine, alanine, and lysine than milk (Frias et al. 2017).
Additionally, fermentation produces vitamins not otherwise found in milk such as vitamins K2, B1, B2 and folic acid (Kim and Oh 2013; Linares et al. 2017).
Many of the benefits associated with kefir are due to the probiotics resulting from the fermentation process. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Sanders 2008).
Oftentimes, the mechanism by which a probiotic microorganism acts in the body to influence health is not well-understood. It is known, however, that certain metabolites produced by microorganisms in fermented foods, like bacteriocins and organic acids, can promote health beyond basic nutrition. Dallas et al. (2016), for example, reported that the microorganisms in kefir grains substantially digest milk proteins, releasing 609 peptides,
25 of which were identified from the literature as having antihypertensive, antimicrobial, immunomodulatory, opioid and/or anti-oxidative properties.
Other health benefits associated with kefir are attributed to the nutraceutical properties of kefiran, the exopolysaccharide (EPS) that forms kefir grain walls (Stepaniak and Fetliński 2002). Kefiran has been shown to be antimicrobial (Rodrigues et al. 2005a;
Medrano et al. 2008), antihypertensive (Maeda et al. 2004), and immunostimulatory
(Vinderola et al. 2005; Medrano et al. 2011).
2.2 Clostridioides difficile Infection
2.2.1 Background
Clostridioides difficile Infection (CDI) is the most common hospital-acquired infection in the United States, and is classified as an urgent threat to public health by the
19
Centers for Disease Control and Prevention (Lewis et al. 2017). Approximately half a million people are infected with Clostridioides difficile Infection (CDI) each year in the
US, and annual CDI related expenses total an estimated US$6.3 billion (Zhang et al.
2016; Mills et al. 2018). Of those infected, 29,000 die within 30 days and approximately half of the deaths are directly attributable to CDI.
Clostridioides difficile is a gram-positive, spore-forming, anaerobic bacillus. In the vegetative form, C. difficile bacteria produce toxins that cause mucosal damage and inflammation, leading to pseudomembranous colitis and diarrhea (Kelly et al. 1994).
Most commonly, patients contract CDI in the hospital, when they are on antibiotics for another ailment (Caroff et al. 2017). In a healthy intestine, the indigenous microflora resist colonization of foreign bacteria like C. difficile, meaning the pathogen is not able to colonize the gut (Wilson 1993). When a patient takes a course of antibiotics, however, the microflora of the intestine is disrupted, which gives C. difficile spores the opportunity to colonize. Although any antibiotic may induce changes to the gut microflora that lead to
C. difficile colonization, patients treated with broad-spectrum antibiotics are the most susceptible (Garey et al. 2008). Exposure to C. difficile occurs by the oral-fecal route.
Patients with CDI shed C. difficile bacteria in their stool, and from there it can contaminate the surrounding environment including toilets, floors, bedding, and furniture.
Since C. difficile spores are resistant to heat, alcohol-based cleaning agents, and quaternary ammonium compounds, it is very difficult to eradicate from hospitals and long-term care facilities where it is present (Mills et al. 2018). Thus, other hospital patients inadvertently ingest C. difficile spores. These spores may then colonize the gut
20 where they convert to vegetative form and produce toxins. Roughly 25% of adults who recently took antibiotics are colonized with C. difficile, but many do not show symptoms of the illness (Kelly et al. 1994).
Patients typically present with CDI 5-10 days after beginning antibiotic treatment.
However, infection may occur up to 8 weeks later. Symptoms of CDI are mild to severe watery diarrhea, blood or pus in the stool, abdominal cramping, fever, nausea, dehydration, and rapid heart rate (Kelly et al. 1994). Severe cases of CDI may be life- threatening.
2.2.2 Treatment Methods
A patient’s first incidence of CDI is treated with orally administered antibiotics such as metronidazole, or vancomycin for those who cannot tolerate metronidazole
(Teasley et al. 1983; Bricker et al. 2005). Oral metronidazole and vancomycin have similar effectiveness for treating CDI, but each has disadvantages. Some strains of C. difficile are resistant to metronidazole and it causes side effects including nausea, vomiting, loss of appetite, headache, and metallic taste. The primary drawback of vancomycin, in contrast, is the high cost. In most cases, initial treatment with oral antibiotics successfully cures CDI. However, in 20-30% of cases the treatment is not effective and the patient relapses one to three weeks after they complete the course of antibiotics (Gerding et al. 2008; Cole and Stahl 2015). Upon relapse, another course of antibiotics is typically prescribed (Table 4, attributed to Cole and Stahl (2015)).
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Patient presentation Treatment options Asympotmatic carrier No treatment Initial episode or first recurrence Discontinue inciting antibiosis Mild disease Metronidazole 500 mg PO q 8 hr x 10-14 d Moderate-severe disease Vancomycin 125 mg PO q 6 hr x 10-14 d Severe or complicated disease Vancomycin 500 mg PO q 6 hr and metronidazole 500 mg IV q 8 hr, and vancomycin enema q 6 hr Second recurrence Tapered/pulsed vancomycin: Vancomycin 125 mg PO q 6 hrs x 10-14 d, then q 12 hr x 7 d, then qd x 7 d, then qod x 8 d, then once every 3 x 15 d Third recurrence Fecal microbiota therapy or Fidaxomycin 200 mg PO q 12 hr x 10 d or IVIg Table 4. CDI Treatment Strategies. Abbreviations: d, day; hr, hours, PO, per os (oral); q, every; qd, daily; qod, every other day. (Cole and Stahl 2015).
If the patient continues to relapse after discontinuing antibiotics, they have what is called a recurrent infection. Since the C. difficile bacteria infecting a patient with recurrent CDI are difficult to eradicate with conventional means, other treatment methods must be employed. To date, one of the most successful alternative treatments for recurrent CDI is fecal microbiota transplantation (FMT). In a fecal microbiota transplantation, intestinal microorganisms are taken from the stool of a healthy donor and transplanted into the colon of the infected individual by oral capsule or colonoscopy. The aim of FMT therapy is to recolonize the gut with protective microorganisms that were destroyed by the use of antibiotics (van Beurden et al. 2017). These microorganisms disrupt both ongoing replication/colonization and toxin production of C. difficile, thereby curing the patient of CDI.
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FMT has been shown to be an effective treatment for CDI in approximately 85% of cases (Drekonja et al. 2015; Spinler et al. 2016). However, it is classified as an investigational therapy by the US Food and Drug Administration (McKinney 2013), so the procedure may not be covered by health insurance in the U.S.. In addition, the long- term risks of FMT are still unknown. In humans, it has been found that obesity may be transferred from the donor to the recipient (Alang and Kelly 2015; van Beurden et al.
2017), and animal studies have demonstrated transference of high blood pressure and atherosclerosis from FMT (Spinler et al. 2016). A less expensive and non-invasive method for restoring the gut microbiota of patients with CDI is to administer probiotics orally in capsule form or in food (Goldstein et al. 2017; Lewis et al. 2017).
Saccharomyces boulardii, for example, has been shown to inhibit C. difficile toxins in the human gut (Castagliuolo et al. 1999; Chen et al. 2006).
2.2.3 Kefir and Clostridioides difficile Infection
More recently, various groups have studied the effect of potentially probiotic strains isolated from kefir on the virulence of C. difficile. In particular, Lactobacillus kefir strains isolated from kefir have been found to impede the action of C. difficile toxins on eukaryotic cells in vitro (Carasi et al. 2012; Bolla et al. 2013). To date, limited studies have examined the effects of kefir probiotics on recurrent CDI in vivo, and the results are conflicting. In one study, 84% of patients who supplemented a six-week Staggered-and-
Tapered-Antibiotic-Withdrawal (STAW) therapy with regular ingestion of commercially- manufactured kefir successfully resolved their recurrent CDI-associated diarrhea for at least nine months (Bakken 2014). On the contrary, another study that followed the
23 protocol set forth by Bakken for treatment of recurrent CDI in a murine model found that supplementation with commercial kefir actually worsened the disease state of infected mice (Spinler et al. 2016). However, in this study, the authors themselves conceded to the limitations of animal models for predicting the outcomes of the same treatment in humans. Even without the opposing evidence presented by Spinler et al., the study by
Bakken (2014) is lacking for a number of reasons including sample size and experimental design flaws. In view of the inconsistencies and limitations of the existing literature, there is a need for further study of the impact of kefir consumption on recurrent CDI.
2.3 Kefir Post-Manufacture Processing
There are also more practical issues pertaining to the use of commercial kefir for treatment of recurrent CDI. The protocol set forth by Bakken required the consumption of
15 ounces of kefir a day for at least 14 weeks (Bakken 2014). This is a large volume of kefir to expect patients to ingest, as it is acidic and has a strong fermented flavor. Despite the known health benefits of kefir, flavor is still the number one attribute that determines consumption of a fermented dairy product (Lightspeed/Mintel 2018). Thus, acceptability of a kefir product is likely to play an important role in patient adherence to prescribed dosages.
In order to improve acceptability, kefir may be further processed using lyophilization or vacuum evaporation to remove undesirable volatile organic compounds
(VOCs) that greatly contribute to flavor (Cheng 2010). However, heat during processing must be minimized to avoid reducing the population of beneficial microorganisms.
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2.3.1 Application of Vacuum Drying Technologies for Removal of VOCs
Volatile organic compounds (VOCs) are a group of low molecular weight aliphatic and aromatic compounds that have a high vapor pressure at room temperature
(Fleming-Jones and Smith 2003). VOCs are responsible for odor or aroma of foods. Due to their low boiling point, VOCs easily change phase from solid or liquid, to gas. As a gas, VOCS can be detected by the olfactory system and perceived as smell. Aroma is an important factor that contributes to overall consumer acceptance of a food or beverage.
Although traditionally used as drying technologies, freeze-drying and vacuum evaporation both have the potential to be used as technologies for removal of VOCs in kefir and other products in which reduction of undesirable volatiles is desirable. Many
VOCs have high boiling points at atmospheric pressure. The low pressures used in freeze-drying and vacuum evaporation, however, reduce the boiling point of volatile compounds. Thus, drying technologies that use vacuum could be applied to strip VOCs that contribute to undesirable flavor in kefir. Another important advantage of applying vacuum drying technologies to kefir is that the low operating temperatures used are favorable for preserving the live cultures present in kefir.
2.3.2 Lyophilization
Lyophilization, or freeze-drying, is a drying method in which water is removed from a frozen product by sublimation (primary drying) and desorption (secondary drying)
(Nireesha et al. 2013). Sublimation or transition of a substance directly from the solid
25 phase to the gas phase without passing through the liquid stage, is achieved in freeze- drying by the use of vacuum.
Figure 5, attributed to Knudsen (2011) shows the phase diagram of water, including the triple point (PTP). The triple point (eutectic point) of water, or the temperature and pressure at which the solid, liquid and gas phase coexist in thermodynamic equilibrium, occurs at a minimum pressure of 4.579 mm Hg (0.0063 atm) and a minimum temperature of 0.0099°C (Nireesha et al. 2013). Water in systems held below the eutectic point may sublimate.
Figure 5. Phase diagram of water (Knudsen 2011).
The freeze-drying technique consists of four fundamental components: freezing, vacuum, heating, and condensation. The product to be dried must first be frozen, usually under atmospheric conditions and to a final temperature of approximately -45°C. Free
26 water in the product will be frozen into the crystalline state, while bound water will enter into the glassy state (Figure 6, attributed to Roos (2010)).
After freezing, the product must be placed under vacuum sufficient to bring the product below the eutectic point. Next, heat is applied to accelerate drying. Sublimation
(primary drying) occurs first. Once all of the water present in the crystalline state is removed from the product, water in the glassy state may be removed via desorption
(secondary drying). Water vapor removed from the product is condensed on the surfaces of a condenser. The condenser is held at a temperature below the product temperature, typically -40 to -80°C, in order to maintain a concentration gradient of water vapor between the drying chamber and the condenser. This concentration gradient is the driving force for water removal in freeze-drying.
27
Figure 6. State diagram of sucrose solution demonstrating the onset of the glass transition, Tg, in maximally freeze-concentrated solution (Roos 2010).
The advantages of lyophilization as a drying technique are that the application of vacuum allows processing temperatures to remain low, which reduces chemical and microbiological changes in the product. In addition, freeze-drying is relatively easy and requires minimal handling of product. The primary disadvantages of freeze-drying are the cost due to the high energy inputs required, and the time needed to dry.
2.3.3 Vacuum Evaporation
Vacuum evaporation is a concentration technique commonly used in the dairy industry to reduce the moisture content of liquid products, usually as a pretreatment before drying processes. The use of partial vacuum lowers the boiling point of water, and
28 thus, the required operating temperature. As a result, damage to the product caused by heat during processing is minimized.
There are two main types of vacuum evaporators: batch evaporators (vacuum pan) and falling film evaporators. In the batch evaporator, product is heated with indirect steam in a jacketed kettle. Water vapor removed from the product is exhausted to a condenser that is connected to a vacuum pump. The major advantage of batch evaporators is their simplicity, while disadvantages include poor heat transfer and long residence times. In a falling film evaporator, product falls due to gravity in a thin film down a long tube, where it is indirectly heated by steam. Water vapor is removed from the system via an exhaust line, and is carried to a condenser. The advantages of falling film evaporators are the short residence times and the ability to operate continuously, while the primary disadvantage is cost of the equipment.
2.4 Methods of Analyses: Selected-Ion Flow-Tube Mass Spectrometry
Selected-ion flow-tube mass spectrometry (SIFT-MS) is a quantitative technique for real-time trace gas analysis with a detection limit at a parts-per-trillion (ppt) level. In
SIFT-MS, volatile organic compounds (VOCs) present in air or in sample headspace are introduced into a flow-tube where they are carried by fast-flowing helium gas and are
+ + chemically ionized by H3O , NO , and O2+ (reagent ions) that are introduced into the helium at a controlled rate (Španěl and Smith 2007). These reagent ions do not react with the major components of air (N2, O2, Ar), or with CO2 or water vapor, allowing trace-gas analysis without the need for preconcentration (Smith and Španěl 2005). After the sample headspace is introduced to the flow-tube, the analyte volatile compounds and reagent ions
29 are allowed to react for a defined period of time. Then, the products of the reaction and the remaining reagent ions are detected by a downstream mass spectrometer. The concentrations of trace gas compounds in the sample are calculated using the mass spectrometer data, the known rate coefficient and a SIFT-MS compound library, which lists the product ions for the reaction of a compound with a given reagent ion (Španěl and
Smith 2007, 2011).
SIFT-MS has many advantages as an analytical technique for quantification of trace gases. To begin, SIFT-MS offers high sensitivity, high selectivity, real-time analysis, concurrent analysis of a mixture of VOCs, and absolute quantification of trace gases. In addition, SIFT-MS requires no or minimal sample preparation and calibration with chemical standards is not necessary. Finally, SIFT-MS is capable of distinguishing between isobaric compounds as well as isomers for some classes of organic compounds.
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Chapter 3: Materials and Methods
3.1 Stock Solution Preparation
Seventy-two 32-oz bottles of plain, unsweetened commercial kefir with 1% milkfat were used for stock solutions and were purchased from local supermarkets
(Columbus, OH). Three 256-oz stock solutions of kefir served as three true replicates
(three trials) throughout the entire study. Each stock solution was prepared by mixing together for 1 minute twenty-four 32-oz bottles (256 oz) of commercial kefir using a
Breddo Likwifier™ 18-gallon industrial liquefier (Breddo Food Products Corporation,
Kansas City, KS). After mixing, the stock solutions were stored at 4°C in sanitized, food grade plastic buckets with lids.
3.2 Sample Preparation
3.2.1 Control Samples
For the untreated control samples, 128 ounces of kefir were removed from each stock solution and stored at 4°C in a sanitized, food grade plastic bucket with a lid for 48 hours before analysis.
3.2.2 Vacuum Evaporation
For the vacuum evaporated samples, a 320-oz aliquot from the stock solution was vacuum evaporated using an SPX Anydro laboratory vacuum evaporator (SPX Flow
Technology Danmark A/S, Soeberg, Denmark). Three independent runs of the evaporator were conducted, one for each stock solution. For each run, product was manually poured
31 into the evaporator calandria, and then the vacuum was set to -0.8 ± 0.04 bar (159,950 ±
30,000 mTorr). Product was heated with indirect steam to a temperature of 43 ± 2°C and was circulated through the evaporator for 20 minutes at this temperature. After evaporation, the product was reconstituted in a sanitized, food grade plastic bucket with deionized water to achieve a moisture content equal to the control samples (10.5% total solids). Reconstituted samples were blended with a Cuisinart® Smart Stick Hand Blender
(Cuisinart, Stamford, CT) for one minute to ensure sufficient rehydration and homogeneity. After reconstitution, vacuum evaporated samples were packaged into sanitized, food grade plastic buckets with lids and were stored at 4°C for 48 hours before analysis.
3.2.3 Lyophilization
For the lyophilized samples, three 64-oz aliquots from the stock solution were loaded into individual trays (64-oz per tray) of a VirTis Ultra 35LE pilot lyophilizer (SP
Scientific, Warminster, PA). Product from all three stock solutions (A, B, C) was lyophilized in the same run, and tray placement on the 9 lyophilizer shelves was randomized, as shown in Figure 7.
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Figure 7. Randomized placement of 9 kefir samples in lyophilizer. A: Stock A, B: Stock
B, C: Stock C.
Once sample was loaded into the trays, the lyophilizer door was sealed and the freezer and condenser were activated. Product was cooled for approximately 24 hours, until the center of the product in each tray reached a temperature of -40°C or below.
Next, the vacuum pump was turned on. Once a pressure ≤100 mTorr was achieved, heat was activated, and shelf temperature was increased at a rate of approximately 1°C/hour until a final moisture content of ≤ 7% had been achieved. The drying time took approximately 72 hours, and final temperature of the product was 20°C.
33
Dried product was removed from the lyophilizer, and product from all three trays pertaining to one stock solution was mixed together in a sanitized, food grade plastic bucket with a lid. Next, dried product was reconstituted with deionized water to achieve a moisture content equal to the control samples (10.5% total solids). Reconstituted samples were blended with a Cuisinart® Smart Stick Hand Blender (Cuisinart, Stamford, CT) for one minute to ensure sufficient rehydration and homogeneity. After reconstitution, lyophilized samples were packaged into sanitized food grade plastic buckets with lids and were stored at 4°C for 48 hours before analysis.
3.3 Methods of Analysis
3.3.1 Moisture Analysis
The moisture content of samples (treated and untreated) was measured using a
CEM SMART 6™ (CEM Corporation, Matthews, NC) according to AOAC official
Method PVM 1:2004 (Mcmanus et al. 2004). The moisture content of samples was measured before and after reconstitution, in the case of vacuum evaporated and lyophilized samples. All measurements were performed in triplicate.
3.3.2 Selected-Ion Flow-Tube Mass Spectrometry
3.3.2.1 Identification of Potential Kefir Compounds
Sample headspace volatile organic compounds (VOCs) were monitored using selected-ion flow-tube mass spectrometry (SIFT-MS) (V200 SYFT Technologies,
Christchurch, New Zealand). The analysis method was previously developed by our group using a full mass scan (FMS) that identified potential compounds in kefir. The 27 compounds selected for the method were chosen based on published literature. Table 5
34 summarizes the kinetic data of the volatile compounds including their corresponding ion products, mass-to-charge ratios (m/z), the precursor or reagent ions, and the reaction rates used in the selected ion mode (SIM) scan method of analysis in the SIFT-MS.
Compound Reagent/ Product Ion m/z+ Reaction rate, k Precursor (x10-9 cm3 s-1) Ion + + 2,3-butanedione H3O C4H7O2 87 1.7 + + NO C4H6O2 86 1.4
2-methyl-1- NO+ C H O+ 87 2. 4 butanol 5 11
2-methylbutanoic O + C H O+ 74 1.8 acid 2 4 10
+ + 2-methylfuran H3O C5H6O.H 83 3.0
+ + 2-pentanone NO NO .C5H10O 116 3.1
+ + 2-phenylethanol NO C8H10O 122 2.3
+ + Acetaldehyde H3O C2H5O 45 3.7
+ + Acetic acid H3O CH3COOH2 61 2.6 + CH3COOH2 • H2O 79 2.6 + + NO NO • CH3COOH 90 9.0
+ + Acetone H3O C3H7O 59 3.9
+ + Benzene H3O C6H6.H 79 1.9 Continued
Table 5. Kinetics of the volatile compounds measured using the selected ion mode (SIM) scan method of the SIFT-MS.
35
Table 5 continued
Compound Reagent/ Product Ion m/z+ Reaction rate, k Precursor (x10-9 cm3 s-1) Ion + + Butanoic acid H3O C3H7COOH2 89 3.0 + C3H7COOH2 • H2O 107 3.0
+ + Butanone NO NO .C4H8O 102 2.8
+ + Carbon disulfide H3O CS2.H 77 1.3 + + O2 CS2 76 4.0 + + Dimethylsulfide H3O (CH3)2S.H 63 2.5
+ + Dimethylsulfone NO C2H6O2S.NO 124 1.4 + + O2 C2H6O2S 94 1.6 + C2H7O2S 95 1.6
+ + Ethanol NO C2H5O 45 1.2 + C2H5O • H2O 63
+ + Ethyl acetate NO NO .CH3COOC2H5 118 2.7
+ + Ethyl butanoate H3O C6H12O2.H 117 3.0 + C6H12O2.H • H2O 135
+ + Ethyl hexanoate H3O C8H16O2.H 145 3.0 + C8H16O2.H • H2O 163
+ + Formic acid O2 HCOO 45 2.0
+ + Isoamyl acetate H3O C7H15O2 131 3.1 + C7H15O2 • H2O 149 + + NO C7H14O2.NO 160 2.4
+ + Methanol H3O CH5O 33 2.7 + CH3OH2 • H2O 51
+ + Methional H3O C4H8OS.H 105 3.0 Continued
36
Table 5 continued
Compound Reagent/ Product Ion m/z+ Reaction rate, Precursor Ion k (x10-9 cm3 s-1) + + Pentanal H3O C5H11O 65 3.6
+ + Pentanoic acid H3O C4H9COOH2 103 2.9 + C4H9COOH2 • 121 H2O
+ + Propanal O2 C3H6O 58 3.1
+ + Pyrazine H3O C4H4N2.H 81 3.4
3.3.2.2 Sample Preparation and SIFT-MS Analysis
Before samples were analyzed, the instrument was validated using a pressurized mixture of certified gas standards (benzene, ethylene, isobutane, octafluorotoluene, hexafluorobenzene, toluene, p-xylene, and 1,2,3,4-tetrafluorobenzene) each having a concentration of 2 ppm (±5%) in nitrogen (Air Liquide America Specialty Gases LLC,
Plumsteadville, PA) and regulated to a pressure of 21 kPa (3 psi). Ultra-high purity helium (UHP 99.999%, Praxair, Columbus, OH) regulated to a pressure of 170 kPa (25 psi) was used to thermalize reagent ions and act as the carrier gas. To prepare samples for analysis, 5 g of sample was placed in an oven-baked 100 mL Pyrex® reagent bottle, capped with a Teflon® septum, and heated in a water bath at 40°C for 30 minutes to allow for headspace equilibration (Taylor et al. 2013). After heating, the sample was presented to the SIFT-MS instrument by inserting a passivated sampling needle (3.8 cm)
+ into the septum, and sample headspace was analyzed using 3 ionizing reagent ions (H3O ,
+ + -1 NO , O2 ). The sample inlet flow was normalized to a rate of 0.346 ± 0.014 Torr∙L s or
37
26 ± 1 sccm (standard cubic centimeters per minute) under standard state ambient temperature (298 K) and pressure (1 Bar). An air scan (blank) was analyzed in between samples to minimize carry-over effects. All measurements were performed using 5 replicates.
3.3.3 Sensory Evaluation
Sensory evaluation for experimental product was conducted using 64 untrained panelists (21 male and 43 female), ages 40-75. A modified group of experimental vocabulary terms for profiling kefir as described by Muir et al. (1999) was used for sensory evaluation. Panelists evaluated overall likeability, overall appearance, acid/sour aroma, creamy/buttery aroma, yeasty/bready aroma, acid/sour flavor, creamy/buttery flavor, green apple flavor, vinegar flavor, and aftertaste using a nine-point hedonic scale
(1=dislike extremely, 5= neither like nor dislike, 9=like extremely). Acid/sour flavor intensity was also evaluated using a five-point just-about-right (JAR) scale (1=much too weak, 3=just about right, 5=much too strong). Panelists were also asked to comment on what they liked and disliked about each sample.
Samples from each stock (A, B, and C) and each treatment group (control, lyophilized, and vacuum evaporated) were presorted by 5 members of our group. It was determined that there were no conspicuous differences between stocks for samples corresponding to the same treatment group. Thus, in order to not overload panelists with samples, all samples used for sensory evaluation came from treatments corresponding to stock C. Panelists were served one 1-ounce sample for each treatment (control, lyophilized, and vacuum evaporated). All samples were evaluated after 48 hours of 38 storage at 4°C. Samples were served at 4°C in small plastic cups labeled with a three- digit random number. Serving order followed a William’s design latin square that was balanced for first order carryover effects (Williams 1949).
3.3.4 Enumeration of Viable Microorganisms
To evaluate the effect of the experimental evaporative processing treatments on the microbial load of the product, control, lyophilized, and vacuum evaporated samples were spread plated on Difco™ Lactobacilli MRS agar (Becton Dickinson, Franklin
Lakes, NJ) with 20 mg/L bromocresol green (Fisher Scientific, Hampton, NH), M-17
Lactococcus agar (Sigma-Aldrich, St. Louis, MO) with 20 mg/L bromocresol green and
50 mL/L 10% lactose solution (Hilmar Ingredients, Hilmar, CA), and Difco™ Potato
Dextrose agar (Becton Dickinson, Franklin Lakes, NJ) acidified to pH 3.5 with 10% tartaric acid solution (Fisher Scientific, Hampton, NH). All media was sterilized before use by autoclaving at 121°C for 20 minutes. A 10 g aliquot of sample was diluted with 90 mL 0.1% Bacto ™ peptone salt solution (Becton Dickinson, Franklin Lakes, NJ) and massaged by hand for 1 minute to mix. Then, sample was serially diluted in 0.1% peptone salt solution and 0.1 mL portions of the respective dilutions were spread plated.
MRS and M-17 agar plates were incubated at 29°C for 2 days in an anaerobic atmosphere achieved with a candle jar. PDA plates were incubated at 25°C in aerobic conditions for 5 days. All samples were plated in duplicate. Results were expressed as colony-forming units (CFU) per milliliter of kefir.
39
3.3.5 Fermentation Rate
In order to determine the ability of cultures present in control, lyophilized, and vacuum evaporated samples to ferment, UHT milk with 2% milkfat (Dean Foods, Dallas,
TX) was inoculated with experimental product at a rate of 4% (v/v) and was incubated at
29°C for 12 hours. Sample pH was measured every 2 hours with a SevenCompact™ pH meter (Mettler Toledo, Columbus, OH). A single fermentation rate analysis was performed for each sample.
3.3.6 Statistical Analysis
Data were fitted to a linear mixed model using RStudio version 1.2.1335
(RStudio, Inc., Boston, MA). The fixed effect was treatment for all analyses. For SIFT-
MS, enumeration of microorganisms, and fermentation rate datasets the random effect was stock. For sensory evaluation datasets the random effect was panelist. A Wald test was used to determine significant differences between treatments (P < 0.05).
40
Chapter 4: Results and Discussion
4.1 Selected-Ion Flow-Tube Mass Spectrometry
Sample headspace volatile organic compounds (VOCs) were monitored using selected-ion flow-tube mass spectrometry to assess how well each treatment separated volatile compounds that contribute to kefir aroma and flavor. The analysis method scanned for 27 compounds, which are listed in Table 6.
The vacuum evaporation and lyophilization treatments significantly reduced the concentration of all of the measured compounds, except for isoamyl acetate (Appendix).
The concentration of isoamyl acetate in the control was negligible, however.
It was expected that vacuum evaporation would remove more VOCs than lyophilization due to the higher temperatures used in vacuum evaporation. However, vacuum evaporation and lyophilization were equally effective in reducing kefir volatiles.
Vacuum evaporation had a significantly greater effect on compound concentration for 10 compounds, whereas lyophilization had a significantly greater effect on compound concentration for 7 compounds. There was no significant difference between treatments for 10 compounds. Considering the effect of the experimental treatments on total volume of the measured VOCs, vacuum evaporation reduced cumulative VOC concentration by
61.63%, compared to lyophilization which reduced cumulative VOC concentration by
61.51%.
41
Compound Odor Descriptor(s) Boiling Point (°C) at 760 mmHg Alcohols 2-Methyl-1-butanol Roasted, wine, onion 127.5 2-Phenylethanol Sweet, floral, fresh, bready 218.2 Ethanol Dry, dust 78.2 Methanol Sweet, pungent 64.7 Aldehydes Acetaldehyde Pungent, fruity, musty 20.1 Methional Yeasty, bready, tomato 165.5 Pentanal Pungent, almond-like, apple 103.0 Propanal Pungent, fruity 48.0 Carboxylic acids 2-Methylbutanoic acid Butter, cheese, sour 176.5 Acetic acid Vinegar, peppers, sour 117.9 Butanoic acid Penetrating, rancid 163.7 Formic acid Pungent, penetrating 101.0 Pentanoic acid Penetrating, rancid 186.1 Esters Ethyl acetate Pineapple, fruity, apple 77.1 Ethyl butanoate Buttery, pineapple, banana 121.5 Ethyl hexanoate Young cheese, pineapple 167.0 Isoamyl acetate Sweet, fruity, banana, pear 142.5 Furans 2-Methylfuran Chocolate 65.0
Heterocylic aromatic compounds Pyrazine Nutty, corn 115.0 Hydrocarbons Benzene Petroleum 80.0 Continued
Table 6. Volatile compounds measured using the selected ion mode (SIM) scan method of the SIFT-MS and their odor descriptors and boiling points at atmospheric pressure
(Walsh et al. 2016; Kim et al. 2019).
42
Table 6 continued
Compound Odor Descriptor(s) Boiling Point (°C) at 760 mmHg Ketones 2,3-Butanedione Buttery 88.0 2-Pentanone Orange peel, sweet, fruity 102.2 Acetone Earthy, fruity, wood pulp 55.8 Butanone Sweet, sharp, butterscotch 79.5 Sulfur compounds Carbon disulfide Sweet, ethereal 46.0 Dimethyl sulfide Cabbage, radish 37.3 Dimethyl sulfone Sulfurous, hot, milk 238.0
The degree to which each volatile compound was removed from kefir can in part be explained by relative volatility. Relative volatility (α) of component A with respect to component B is defined as follows:
° 푝 푥 푃 훼 , = = ° 푝 푥 푃
where p is the partial pressure, x is the mole fraction in liquid, and P° is the vapor pressure. In this case, A would be the volatile compound, and B would be the kefir. The larger the value of α above 1.0, the easier the separation of the volatile from kefir. Kefir is a complex mixture and so the relative volatilities of the studied VOCs in kefir cannot be calculated. Nevertheless, in theory, compounds with high relative volatility would have been separated from kefir to a greater extent than compounds with low relative volatility during the drying processes.
43
It is clear, however, that relative volatility was not the only factor governing separation of VOCs from kefir. If the degree of separation of each VOC was based solely on relative volatility, then one experimental treatment should have been consistently better at removing volatile compounds. Instead, vacuum evaporation was more effective in reducing certain compounds and lyophilization others. A possible reason for this is entrapment of certain volatile compounds in freeze-dried kefir. In freeze-drying, ice crystals that are formed during slow freezing are pure water, meaning aroma compounds remain in the concentrated amorphous phase. Some of these aroma compounds are likely permanently encapsulated by dissolved solids, preventing their separation from the kefir.
Ice crystals formed during fast freezing may also entrap aroma compounds (Coumans et al. 1994). It has also been reported that association between volatile compounds and carbohydrate molecules via hydrogen bonding produced complex structures that controlled volatile permeability (Flink and Karel 1970). Complexation between volatile compounds and macromolecules may also have played a role in volatile removal in vacuum evaporation.
The list of 27 compounds was narrowed down for detailed exploration in the present discussion based on the following criteria: concentration of the compound in control samples was >1000 ppb and at least one of the experimental treatments caused a reduction of >50% in the compound concentration; or at least one of the experimental treatments caused a reduction of >95% in the compound concentration. Compounds fitting these criteria were expected to be key contributors to differences in kefir flavor and acceptability among samples in sensory evaluation. A total of seven compounds fit
44 these criteria: acetaldehyde, acetone, butanoic acid, dimethyl sulfide, ethyl acetate, formic acid, and propanal.
Acetaldehyde is commonly found in milk products, as well as in vinegar, coffee, bread, fruits and vegetables (Uebelacker and Lachenmeier 2011). The odor descriptors of acetaldehyde are pungent, fruity, and musty (Kim et al. 2019). The olfactory threshold of acetaldehyde is 0.21 ppm (Leonardos et al. 1969). Acetaldehyde is a product of carbohydrate metabolism as bacteria and yeast convert pyruvate into acetaldehyde and carbon dioxide during alcohol fermentation.
Acetaldehyde was one of two compounds that had an average concentration greater than 1000 ppb in control samples and was diminished by greater than 95% by one of the experimental treatments. Figure 8 shows the concentration of acetaldehyde in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of acetaldehyde present in the kefir samples. Vacuum evaporation reduced the concentration of acetaldehyde by an average of 96.17%, which is significantly more than the average 51.76% reduction provided by lyophilization.
45
5000
4500
4000
3500
3000
2500 A 2000
1500
1000 C Acetaldehyde Concentration (ppb) Concentration Acetaldehyde 500 B 0 Control Vacuum Evaporated Lyophilized
Figure 8. Concentration of acetaldehyde in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
Acetone is a natural metabolic product of plants and animals. Acetone is produced in the liver from fatty acids, and may pass into milk (Heuer et al. 2001). Thus, acetone naturally occurs in dairy products. The odor descriptors of acetone are earthy, fruity, wood pulp, and hay. The olfactory threshold of acetone is 100.0 ppm (Leonardos et al.
1969). Figure 9 shows the concentration of acetone in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of acetone present in the kefir samples, and there was no significant difference in acetone concentration between treatments. Vacuum evaporation reduced the
46 concentration of acetone by an average 91.20%, while lyophilization reduced the acetone concentration by an average of 87.15%.
5000
4500
4000
3500
3000
2500
2000
1500
Acetone Concentration (ppb) Concentration Acetone 1000 A
500 B B 0 Control Vacuum Evaporated Lyophilized
Figure 9. Concentration of acetone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15). Means with different letters are significantly different (P < 0.05).
Butanoic acid, or butyric acid, is a fatty acid found in animal fat, fluid milk, and other dairy products. As a free fatty acid, butanoic acid has a rancid butter odor (Vaseji et al. 2012; Kim et al. 2019). The olfactory threshold of butanoic acid is 0.001 ppm
(Leonardos et al. 1969). Figure 10 shows the concentration of butanoic acid in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of butanoic acid present in the
47 kefir samples. Lyophilization reduced the concentration of butanoic acid significantly more than vacuum evaporation. Butanoic acid was reduced by an average of 65.46% from lyophilization and by an average of 36.59% from vacuum evaporation.
5000
4500
4000
3500
3000 A
2500
2000 B
1500 C 1000 Butanoic Acid Concentration (ppb) Concentration Acid Butanoic 500
0 Control Vacuum Evaporated Lyophilized
Figure 10. Concentration of butanoic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
Dimethyl sulfide was the second compound, along with acetaldehyde, that had an average concentration greater than 1000 ppb in the control and was diminished by greater than 95% by one of the experimental treatments. Dimethyl sulfide occurs naturally in plants and has a strong cooked-cabbage odor (Kim et al. 2019). Dimethyl sulfide also occurs in fermented dairy products. LAB synthesize dimethyl sulfide from sulfur- 48 containing amino acids (Ibrahim 2011). The olfactory threshold of dimethyl sulfide is
0.001 ppm (Leonardos et al. 1969). Figure 11 shows the concentration of dimethyl sulfide in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of dimethyl sulfide present in the kefir samples. Vacuum evaporation caused a significantly greater reduction of dimethyl sulfide in kefir samples than lyophilization. Vacuum evaporation reduced dimethyl sulfide concentration by an average of 95.21% and lyophilization reduced dimethyl sulfide by an average of 50.62%.
5000
4500
4000 A 3500
3000
2500
2000 C
1500
1000 Dimethyl Sulfide Concentration (ppb) Concentration Sulfide Dimethyl 500 B 0 Control Vacuum Evaporated Lyophilized
Figure 11. Concentration of dimethyl sulfide in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
49
Ethyl acetate is the most common ester in fruits (Dionísio et al. 2012). Ethyl acetate has a distinct fruity aroma and flavor described as similar to pineapple or apple
(Walsh et al. 2016). The olfactory threshold of ethyl acetate is 0.09 ppm (Murnane et al.
2013). Acetic acid bacteria form ethyl acetate from ethanol and acetic acid. Figure 12 shows the concentration of ethyl acetate in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of ethyl acetate present in the kefir samples. Lyophilization reduced the concentration of ethyl acetate by an average of 65.51%, which is significantly more than the average 34.58% reduction provided by vacuum evaporation.
5000
4500
4000
3500
3000
2500
2000 A 1500 B 1000
Ethyl Acetate Concentration (ppb) Concentration Acetate Ethyl C 500
0 Control Vacuum Evaporated Lyophilized
Figure 12. Concentration of ethyl acetate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05). 50
Formic acid has a pungent, penetrating odor similar to vinegar, and a sour taste
(Kim et al. 2019). The odor threshold of formic acid is 0.52 ppm (Murnane et al. 2013).
Formic acid occurs naturally in fruits, honey, and in fermented foods. Formic acid is a product of carbohydrate metabolism. Heterofermentative bacteria ferment glucose to acetic acid, ethanol, formic acid, and CO2 (Jurtshuk 1996). Figure 13 shows the concentration of formic acid in control, vacuum evaporated, and lyophilized samples.
Both vacuum evaporation and lyophilization significantly decreased the concentration of formic acid present in the kefir samples. There was a significantly greater decrease in the mean concentration of formic acid for the lyophilized treatment group than for the vacuum evaporated group. Lyophilization reduced formic acid concentration by an average of 65.23% and vacuum evaporation reduced formic acid by an average of
52.41%.
51
5000
4500
4000
3500
3000 A
2500
2000
1500 B C 1000 Formic Acid Concentration (ppb) Concentration Acid Formic 500
0 Control Vacuum Evaporated Lyophilized
Figure 13. Concentration of formic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
Propanal, or propionaldehyde, occurs naturally in various fruits, bread, cheese, and coffee. In fermented dairy, propanal is a product of lipid metabolism (Walsh et al.
2016). The odor threshold of propanal is 0.0009 ppm (Murnane et al. 2013). Figure 14 shows the concentration of propanal in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of propanal present in the kefir samples. There was no significant difference in propanal concentration between treatments. Vacuum evaporation reduced propanal concentration by an average of 65.23% and lyophilization reduced propanal by an average of 52.41%.
52
5000
4500
4000
3500
3000
2500
2000
1500 A
Propanal Concentration (ppb) Concentration Propanal 1000
500 B B 0 Control Vacuum Evaporated Lyophilized
Figure 14. Concentration of propanal in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
In summary, vacuum evaporation subjected kefir to a temperature of 43 ± 2°C and pressures slightly below atmospheric pressure, whereas lyophilization subjected kefir to temperatures between -40°C – 20°C and pressures well below atmospheric pressure.
Despite the differences in processing conditions, the temperature, pressure, and time supplied by both the vacuum evaporation and lyophilization treatments was sufficient to cause a significant decrease in the concentration of 26 of the 27 kefir VOCs that were monitored using SIFT-MS. The vacuum evaporation and lyophilization treatments were equally effective in removing VOCs from kefir, as both treatments reduced cumulative 53
VOC concentration by approximately 62%. Despite the overall similarity between treatments, each process separated certain compounds to a greater extent than the other, which may be explained by microencapsulation of volatile compounds by solids dissolved in the amorphous phase of lyophilized kefir.
4.2 Sensory Evaluation
Sensory evaluation for experimental product was conducted using 64 untrained panelists to determine effect of vacuum evaporation and lyophilization on kefir aroma, flavor, and acceptance. Panelists rated overall likeability, overall appearance, acid/sour aroma, creamy/buttery aroma, yeasty/bready aroma, acid/sour flavor, creamy/buttery flavor, green apple flavor, vinegar flavor, and aftertaste using a nine-point hedonic scale
(1=dislike extremely, 5= neither like nor dislike, 9=like extremely). Figure 15 shows the mean hedonic scores for kefir sensory attributes. There was no significant difference among treatment groups for any of the kefir sensory attributes. Thus, vacuum evaporation and lyophilization treatments did not significantly improve or worsen the overall acceptability of the kefir. This was not expected as it was predicted that the studied volatile compounds would contribute to overall kefir flavor and aroma, and preliminary
SIFT-MS data showed a difference in VOC concentration between treatments. Informal taste tests among our group also indicated a noticeable difference in the flavor and aroma of the experimental kefir products compared to the control. Future sensory evaluation of vacuum evaporated and lyophilized kefir samples could better elucidate any differences between treatments. For example, a preference ranking test, in which panelists are forced
54 to rank a group of samples with respect to their preference of an attribute, such as overall acceptability, or acid flavor, could reveal whether one or both treatments perform better than untreated kefir. Alternatively, a trained panel may be more capable of describing changes in kefir sensory attributes.
Overall 9 Aftertaste 8 Appearance 7 6 5 Vinegar Flavor 4 Acid/Sour Aroma 3 2 1
Green Apple Flavor Creamy/Buttery Aroma
Creamy/Buttery Flavor Yeasty/Bready Aroma
Acid/Sour Flavor
Control Vacuum Evaporated Lyophilized
Figure 15. Spider plot of sensory attribute hedonic scores for kefir samples (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely). *P < 0.05.
The average rating of each sensory attribute that panelists evaluated was approximately 5, meaning neither like nor dislike, for all three treatment groups.
Although the average score for each sensory attribute was 5 across all treatment groups, tallying the number of responses for each score (1 to 9) revealed that among the panelists,
55 there was a group of people who liked the kefir, a group that disliked the kefir, and a group that was neutral. Figure 16 illustrates the dispersion of panelists in regards to overall acceptability scores of kefir samples.
25
20
15
10 Answers (%) Answers
5
0 123456789
Control Vacuum Evaporated Lyophlized
Figure 16. Histogram distribution of overall acceptability scores for kefir samples (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely).
Along with scoring sensory attributes using a hedonic scale, panelists were also asked to comment on what they liked and disliked about each sample. The most prevalent answer for what panelists liked about samples was the creaminess, while the most prevalent answer for what panelists disliked about the kefir was that it was sour. Similar findings were reported by Muir et al. (1999). Since the mean score for acid/sour aroma
56 and flavor were both approximately 5 for all three samples, it can be assumed that sourness was an important contributor to unacceptance of kefir for the group of panelists who disliked kefir (rather than for those who were neutral or who liked kefir). Of the panelists who disliked the acidity of kefir, many said that the sourness was overwhelming, and that it drowned out any other flavor notes. Thus, although vacuum evaporation and lyophilization substantially reduced the volume of VOCs present in kefir, they did not reduce the sourness of kefir enough to significantly improve kefir acceptability. Sourness in fermented dairy comes primarily from organic acids, especially lactic acid. Lactic acid is a non-volatile compound, and thus was not expected to be removed via vacuum drying processes.
In summary, vacuum evaporation and lyophilization treatments did not significantly improve the liking of kefir sensory attributes among untrained panelists.
Sensory data revealed that there were three distinct groups of consumers: those who tended to like kefir, those who neither liked nor disliked kefir, and those who tended to dislike kefir. Of those who disliked kefir, a primary listed reason was the product sourness. Future work is necessary to better define the changes in kefir sensory profiles caused by vacuum evaporation and lyophilization treatments, and their impact on overall acceptability. In addition, more work is needed to better understand the role of acidity on kefir acceptability.
4.3 Enumeration of Viable Microorganisms
As previously described, samples were spread plated on MRS agar, M-17 agar, and PDA agar to determine the effect of the experimental treatments on the load of
57
Lactobacillus spp., Lactococcus spp, and yeast, respectively, in the product. Although the commercial kefir label claims Saccharomyces florentinus as one of the probiotic cultures present in the product, yeast were not able to be cultured from any of the treatments. As such, this data was excluded from the present discussion. It is likely that yeast are present in the starter culture, but competition for trace nutrients inhibited yeast metabolism resulting in cell death (Narendranath et al. 2001; Bayrock and Ingledew 2004).
Figure 17 shows the concentration of Lactobacillus spp. in control, vacuum evaporated, and lyophilized samples. Both vacuum evaporation and lyophilization significantly decreased the concentration of Lactobacillus spp. present in the kefir samples. However, treated samples still had a high concentration of Lactobacillus spp., with mean values of 108.46 CFU/mL and 108.48 CFU/mL for vacuum evaporated and lyophilized samples, respectively, compared to the control, which had a mean concentration of 108.92 CFU/mL.
Although there is no consensus among scientific communities regarding the minimum concentration of beneficial microorganisms that should be present in products considered probiotic, it is generally accepted that a minimum concentration of 106
CFU/mL is sufficient (Kechagia et al. 2013). Thus, despite the slight decrease in
Lactobacillus spp. caused by the experimental treatments, the treated samples maintain probiotic status. Furthermore, there was no significant difference between sample means for vacuum evaporation and lyophilization treatments.
58
12 11 10 A 9 B B 8 7
CFU/mL) 6 spp. Viable Count Count Viable spp. 10 5
(Log 4 3
Lactobacillus 2 1 0 Control Vacuum Evaporated Lyophilized
Figure 17. Population of Lactobacillus spp. in kefir samples. Data are means and standard errors from three separate trials, with two samples analyzed per treatment (n = 6). Means with different letters are significantly different (P < 0.05).
Figure 18 shows the concentration of Lactococcus spp. in control, vacuum evaporated, and lyophilized samples. As was the case for Lactobacillus spp., both vacuum evaporation and lyophilization significantly decreased the concentration of
Lactococcus spp. present in the kefir samples. However, treated samples still had a high concentration of Lactococcus spp., with mean values of 108.49 CFU/mL and 108.37
CFU/mL for vacuum evaporated and lyophilized samples, respectively, compared to the control, which had a mean concentration of 108.76 CFU/mL. Once again, there was no significant difference between sample means for vacuum evaporation and lyophilization treatments.
59
12 11 10 A 9 B B 8 7 CFU/mL) spp. Viable Count Count Viable spp. 10 6
(Log 5 4
Lactococcus 3 2 1 0 Control Vacuum Evaporated Lyophilized
Figure 18. Population of Lactococcus spp. in kefir samples. Data are means and standard errors from three separate trials, with two samples analyzed per treatment (n = 6). Means with different letters are significantly different (P < 0.05).
It was expected that freeze-drying would substantially reduce the population of
LAB present in the kefir. Freezing and drying both deplete water available to bacteria, resulting in osmotic shock and cell death (Heckly 1985). Additionally, intracellular ice formation during freezing and recrystallization causes membrane injury. Freeze-drying, however, did not substantially reduce the population of Lactobacillus spp. or Lactococcus spp. present in the sample. It is likely that the food matrix in which the bacteria were suspended served as a cryoprotectant, helping to preserve LAB in the product. Skim milk is a widely used cryoprotectant in microbiology (Saarela et al. 2005).
60
The vacuum evaporation treatment was not expected to considerably diminish the population of LAB present in kefir. The conditions for vacuum evaporation were 40°C for 20 minutes. A temperature of 40°C is not high enough to be lethal to LAB. On the contrary, this temperature is capable of causing growth of LAB. Yogurt, which is fermented by Lactobacillus bulgaricus and Streptococcus thermophilus, is incubated at
43°C-46°C. However, growth of LAB in the vacuum evaporated treatment group was unlikely because of the short time that product was held at this temperature.
In summary, vacuum evaporation and lyophilization treatments significantly decreased the concentration of viable LAB present in the kefir samples. LAB are the principal microorganism present in kefir and kefir-isolated LAB have been associated with probiotic effects (Witthuhn et al. 2005; Zheng et al. 2013; Leite et al. 2015). Thus, the concentration of LAB in treated samples is important for overall probiotic quality of the experimental kefir products. As previously discussed, the total number of microorganisms present in a probiotic product should be greater than 106 CFU/mL.
Despite the statistically significant decrease in LAB in the treated kefir samples, the concentration of Lactobacillus spp. and Lactococcus spp. in these samples is still considered sufficient for probiotic products. Thus, the experimental treatments did not diminish the probiotic quality of the product in practical terms. In addition, plate count data indicates that there is no benefit of one treatment over the other in terms of preserving probiotic cultures, since there was no significant difference between sample means for vacuum evaporation and lyophilization treatments.
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4.4 Fermentation Rate
The fermentation rate of UHT milk samples inoculated with 4% (v/v) experimental product was determined to assess the effect of vacuum evaporation and freeze-drying on the activity of live cultures in the product. The fermentation rate of milk inoculated with untreated product (control) served as the baseline. A decrease in fermentation rate compared to the control indicated decreased activity of the microbial population in the product.
Figure 19 shows the fermentation rate of UHT milk inoculated with 4% (v/v) untreated kefir (control), vacuum evaporated kefir, and lyophilized kefir. The mean pH of the untreated kefir (control) group was significantly higher than the corresponding values for vacuum evaporated samples at 4 hours, and significantly lower than corresponding values for vacuum evaporated samples at 6 and 8 hours. By 10 hours, there was no difference between the mean pH of untreated and vacuum evaporated treatment groups.
Considering the general trends of the control and vacuum evaporated curves, the milk inoculated from vacuum evaporated samples seemed to ferment at a slightly slower rate than the control group.
The mean pH of the control group was significantly lower than the corresponding values for lyophilized samples at 2, 6, 8, 10 and 12 hours. Considering the general trends of the control, vacuum evaporated, and lyophilized curves, the milk inoculated from lyophilized samples seemed to ferment at a slower rate than the control group, and at a slightly slower rate than the vacuum evaporated group.
62
6.5 * *
6.25 * * *
6
5.75 * * pH
5.5
5.25 *
5
4.75 0 2 4 6 8 10 12 Time (hrs)
Control Vacuum Evaporated Lyophilized
Figure 19. Fermentation rate of UHT milk inoculated with 4% (v/v) experimental kefir
(untreated (control), vacuum evaporated or lyophilized), incubated at 29°C for 12 hours.
Data are means and standard errors from three separate trails (n=3). Asterisks indicate values that are significantly different (P < 0.05) from corresponding values for untreated samples (control).
63
The fermentation rate data reflects the enumeration of Lactobacillus spp and
Lactococcus spp data. From the plate count data, the combined mean total of LAB present in the samples was 109.15 for the control group, 108.78 for the vacuum evaporated group, and 108.73 for the lyophilized group. It can be assumed that in this assay, fermentation of the milk was a lactic acid fermentation since LAB are the predominant microorganism present in kefir. Milk samples were inoculated at 4% (v/v). Therefore, milk inoculated with vacuum evaporated product was inoculated with fewer lactic acid bacterial cells than milk inoculated with untreated product (control). As a result, milk inoculated with vacuum evaporated product fermented at a slightly slower rate than the control. Similarly, milk inoculated with lyophilized product was inoculated with fewer lactic acid bacterial cells than milk inoculated with vacuum evaporated product. Thus, milk inoculated with lyophilized product fermented at a slightly slower rate than milk inoculated with vacuum evaporated product.
64
Chapter 5: Conclusion
In this study, plain, commercial kefir was subjected to lyophilization or vacuum evaporation treatments. The overall goal of this study was to determine whether the employed vacuum drying techniques could significantly separate VOCs from kefir without affecting the probiotic quality of the product, and whether removal of VOCs would improve the acceptability of kefir. Additionally, the effect of each treatment on kefir VOC concentration, sensory quality, and microbial viability and activity was determined. Loss of volatile compounds was monitored using selected-ion flow-tube mass spectrometry (SIFT-MS); kefir acceptability was evaluated by untrained panelists using a 9-point hedonic scale; microbial viability was determined using selective media for enumeration of Lactobacillus spp. and Lactococcus spp.; and microbial activity was assessed by measuring fermentation rate of milk inoculated with treated and untreated
(control) kefir.
The first objective of this study was to evaluate the efficiency of freeze-drying and vacuum evaporation processes to remove VOCs from kefir. The hypothesis was that vacuum evaporation and freeze-drying would significantly reduce VOCs in kefir due to the low pressures used in the processes, and that vacuum evaporation would separate
VOCs more effectively than freeze-drying due to the higher temperatures used in vacuum evaporation. As was expected, vacuum evaporation and lyophilization treatments
65 significantly reduced VOC content in the kefir. Both experimental treatments caused a significant decrease in the concentration of 26 of the 27 kefir VOCs that were monitored using SIFT-MS, and cumulative VOC concentration was reduced by approximately 62% for both treatments. In contrast to what was expected, there was no significant difference between treatments overall.
The second objective of this study was to investigate whether reducing or eliminating VOCs from kefir enhanced the sensory quality of the product. The hypothesis was that reduction in kefir VOC content would positively impact acceptability of kefir because VOCs play an important role in food aroma and flavor. Sensory evaluation results showed that vacuum evaporation and lyophilization treatments did not significantly improve the liking of commercial kefir. Panelist comments revealed that sourness is an important factor that contributes to consumer dislike of kefir.
The third objective of this study was to determine the effect of lyophilization and vacuum evaporation treatments on kefir microbial content and activity. The hypothesis was that freeze-drying would cause a significant decrease in the concentration of viable microorganisms present in kefir because of low-water stress caused by freezing and drying, and intracellular ice formation; and that vacuum evaporation would not considerably diminish the population of microorganisms in kefir because the operating temperature is not lethal for kefir cultures. Freeze-drying and vacuum evaporation significantly reduced the concentration of Lactobacillus spp. and Lactococcus spp. present in the kefir. Although significant, the population of LAB in the kefir samples was still considered sufficient for probiotic products. Thus, the experimental treatments
66 did not affect the probiotic quality of the kefir in practical terms. Despite there being no significant difference between sample means for vacuum evaporation and lyophilization, milk inoculated with freeze-dried product seemed to ferment at a slightly slower rate than the vacuum evaporated group, which fermented at a slightly slower rate than the control.
The slight differences between fermentation rates among treatment groups may be explained by differences in LAB inoculation levels, as the mean total of LAB present in control samples was slightly higher than in the vacuum evaporated group, which in turn was slightly higher than the lyophilized group.
In conclusion, processing of commercial kefir using two vacuum drying techniques commonly employed by the food-industry, vacuum evaporation and lyophilization, was successful in decreasing the concentration of 26 VOCs without substantially diminishing live LAB that are important for kefir probiotic quality. Thus, vacuum evaporation and freeze-drying have the potential for use as a processing step in kefir production to remove volatile compounds that negatively affect kefir aroma and flavor.
Removal of volatile compounds was predicted to improve kefir acceptability among American consumers; however, in this study sensory evaluation did not show a significant difference between treatments for organoleptic properties of kefir. More work is needed to understand the impact of VOCs on kefir acceptability.
67
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Appendix : Additional Selected-Ion Flow-Tube Mass Spectrometry Results
50
45
40
35
30
25
20
15
10 A 5 B B 2-methylbutanoic Acid Concentration (ppb) Concentration Acid 2-methylbutanoic 0 Control Vacuum Evaporated Lyophilized
Figure 20. Concentration of 2-methylbutanoic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
81
50
45
40
35 A 30
25
20 C
15
10 2-pentanone Concentration (ppb) Concentration 2-pentanone B 5
0 Control Vacuum Evaporated Lyophilized
Figure 21. Concentration of 2-pentanone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
82
50
45
40
35
30
25
20
15
10
2-phenylethanol Concentration (ppb) Concentration 2-phenylethanol 5 A B C 0 Control Vacuum Evaporated Lyophilized
Figure 22. Concentration of 2-phenylethanol in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
83
50
45
40
35
30
25
20
15 A 10 B B Dimethylsulfone Concentration (ppb) Concentration Dimethylsulfone 5
0 Control Vacuum Evaporated Lyophilized
Figure 23. Concentration of dimethylsulfone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
84
50
45
40 A 35
30
25
20 B 15 B
10
Ethyl Butanoate Concentration (ppb) Concentration Butanoate Ethyl 5
0 Control Vacuum Evaporated Lyophilized
Figure 24. Concentration of ethyl butanoate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
85
50
45
40
35
30
25
20
15
10 A
Ethyl Hexaoate Concentration (ppb) Concentration Hexaoate Ethyl B B 5
0 Control Vacuum Evaporated Lyophilized
Figure 25. Concentration of ethyl hexanoate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
86
50
45
40
35
30
25
20
15
10 Isoamyl Acetate Concentration (ppb) Concentration Acetate Isoamyl 5 A A A 0 Control Vacuum Evaporated Lyophilized
Figure 26. Concentration of isoamyl acetate in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
87
50
45 A 40
35
30
25 C 20
15
Pyrazine Concentration (ppb) Concentration Pyrazine 10 B
5
0 Control Vacuum Evaporated Lyophilized
Figure 27. Concentration of pyrazine in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
88
500
450
400
350
300 A
250
200 C
150
100
2,3-butanedione Concentration (ppb) Concentration 2,3-butanedione B 50
0 Control Vacuum Evaporated Lyophilized
Figure 28. Concentration of 2,3-butanedione in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
89
500
450
400
350 A 300
250 B 200
150 C 100
2-methyl-1-butanol Concentration (ppb) Concentration 2-methyl-1-butanol 50
0 Control Vacuum Evaporated Lyophilized
Figure 29. Concentration of 2-methyl-1-butanol in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
90
500
450
400
350
300
250
200
150
100 A
2-methylfuran Concentration (ppb) Concentration 2-methylfuran B 50 B
0 Control Vacuum Evaporated Lyophilized
Figure 30. Concentration of 2-methylfuran in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
91
500
450
400
350
300
250
200
150
Butanone Concentration (ppb) Concentration Butanone 100 A 50 B C 0 Control Vacuum Evaporated Lyophilized
Figure 31. Concentration of butanone in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
92
500
450
400
350
300
250
200
150
100
Carbon Disulfide Concentration (ppb) Concentration Disulfide Carbon A 50 B C 0 Control Vacuum Evaporated Lyophilized
Figure 32. Concentration of carbon disulfide in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n =
15). Means with different letters are significantly different (P < 0.05).
93
500
450
400 A
350
300
250
200 B
150 B
Methanol Concentration (ppb) Concentration Methanol 100
50
0 Control Vacuum Evaporated Lyophilized
Figure 33. Concentration of methanol in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
94
500
450
400
350
300
250 A 200
150 C
Methional Concentration (ppb) Concentration Methional 100
50 B
0 Control Vacuum Evaporated Lyophilized
Figure 34. Concentration of methional in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P
95
500
450
400
350
300 A
250
200 C
150
Pentanal Concentration (ppb) Concentration Pentanal 100 B 50
0 Control Vacuum Evaporated Lyophilized
Figure 35. Concentration of pentanal in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15). Means with different letters are significantly different (P < 0.05).
96
500
450
400
350
300
250
200
150
100 A Pentanoic Acid Concentration (ppb) Concentration Acid Pentanoic 50 B B 0 Control Vacuum Evaporated Lyophilized
Figure 36. Concentration of pentanoic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
97
5000
4500
4000
3500
3000
2500
2000
1500
Acetic Acid Concentration (ppb) Concentration Acid Acetic 1000 A B 500 C
0 Control Vacuum Evaporated Lyophilized
Figure 37. Concentration of acetic acid in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15).
Means with different letters are significantly different (P < 0.05).
98
5000
4500
4000
3500
3000
2500
2000 A 1500 B Benzene Concentration (ppb) Concentration Benzene 1000 C 500
0 Control Vacuum Evaporated Lyophilized
Figure 38. Concentration of benzene in kefir samples. Data are means and standard errors from three separate trials, with five samples analyzed per treatment (n = 15). Means with different letters are significantly different (P < 0.05).
99