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Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

School of Engineering and Sciences

Postbiotic effect evaluation of Lactobacillus acidophilus and

Lactobacillus plantarum 299v lysates obtained through ultrasonication against common skin pathogenic bacteria

A thesis presented by

Blanca Raquel Gutiérrez Prieto

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of

Master of Science In

Biotechnology

Monterrey Nuevo León, Dec 18th, 2020

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Dedication

Dedicated to Neno and Vira, my greatest loves and my forever motivation.

I will always be grateful for having you in my life. .

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Acknowledgements

I would like to thank everyone that trusted me and was part of this two-year experience, specially to my advisor, Dra. Arlette, and committee members, Dr. Pepe and Dr. Benavides, who have supported me since the start. I would also like to include Felipe and Dr. Alberto for sharing their knowledge and friendship with me.

This was definitely one of a kind journey but from all the things that happened, a feeling of satisfaction and happiness remains.

Neno, thank you for always listening to me no matter how repetitive everything I said was. Calling you at the end of the day was one of my favorite things to do so I could recharge my emotional batteries and face life with a smile.

Nena, I want to thank you too for all the support and trust you always give me. For me, it did not matter how often we talked because every time we spoke again, it felt like a big warm hug full of love.

This thesis also goes to Luis, my best friend and life partner. My days were so much better with you by my side. All your jokes, hugs, support and cheering words after stressing all day will always be in my heart. Thanks for the food, too. Sharing a meal with you at the end of the day always brightened me up.

One of the most valuable things I found through this journey is friendship. Boo, chatting at the lab or just waving at you was complete happiness. I will never forget that moment we decided to go for a tiramisu. You are my best friend.

To all my friends that I met through the semesters: Nora, Rebeca, Andrés, Orlando, Gilberto and David, thank you. All the moments we shared are unforgettable.

Finally, I would like to show my deepest appreciation and gratitude to Tecnológico de Monterrey and CONACyT for their support on tuition and living, respectively.

Without their scholarships, all of this would not have been possible.

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V

Postbiotic effect evaluation of Lactobacillus acidophilus and Lactobacillus plantarum 299v lysates obtained through

ultrasonication against common skin pathogenic bacteria by

Blanca Raquel Gutiérrez Prieto

Abstract

The skin microbiome refers to the community of microorganisms that reside on the human skin. There’s an important equilibrium and protection relationship among these microorganisms and the skin that is achieved through metabolite production, immunomodulation and colonization. Microbiota alterations result in dysbiosis, an imbalance in the microbial communities that is associated with the development of cutaneous diseases such as acne vulgaris, atopic dermatitis and impetigo. To recover the skin equilibrium, probiotic bacteria and postbiotic use are attractive approaches because of their role in disease modulation through possible elimination of pathogenic bacteria. Since postbiotics are molecules such as enzymes, proteins and soluble factors produced or released by the probiotic bacteria metabolism, there is less associated risk in comparison with administrating live bacteria through exogenous applications. The objective of this study is to evaluate the antimicrobial effects of postbiotics obtained from L. acidophilus and L. plantarum 299v lysates against the common skin pathogens Cutibacterium acnes, Staphylococcus aureus and Streptococcus pyogenes. Lysates were obtained through ultrasonication (US) alone or a combination and freeze-thawing and US (FT+US). Antimicrobial activity of lysates, supernatants, non-lysed and heat-killed cells was evaluated by the agar well diffusion method and total protein of the lysates was quantified using the Bicinchoninic Acid assay. In this work, results confirm the significant antimicrobial activity of the ultrasonicated lysates of L. acidophilus against S. aureus and C.

acnes, while L. plantarum 299 lysates only showed an effect against C. acnes. Also, the presence of acids is directly correlated with the formation of inhibition zones for

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L. plantarum 299v pH 3.8 supernatant. Lysates result in a complete loss of antimicrobial activity after heating at 80°C for 1.5 h, which suggests a possible denaturalization of antimicrobial peptides. It is concluded L. acidophilus and L.

plantarum 299v postbiotics obtained through cellular lysis using sonication present a strain dependent antimicrobial effect that could be applied to prevent skin diseases such as acne vulgaris and atopic dermatitis.

Keywords: postbiotics, Lactobacillus plantarum, Lactobacillus acidophilus, ultrasonication, antimicrobial activity, skin microbiota, acne vulgaris, atopic

dermatitis.

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Abbreviations

PBS Phosphate-Buffered Saline MRS Man, Rogosa and Sharpe BHI Brain Heart Infusion NL Non-Lysed cells US Ultrasonicated cells

FT+US Freeze-Thawed Ultrasonicated cells HT Heat Treated cells

HT-US Heat Treated-Ultrasonicated cells

HT-FT+US Heat Treated-Freeze Thawed Ultrasonicated cells CFU Colony Forming Units

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List of Figures

Figure 1. Comparison between a healthy skin barrier versus a………..4 damaged skin barrier

Figure 2. Acne vulgaris biogenesis and progression……….……....6 Figure 3. Prebiotic, probiotic and postbiotic characteristics and examples….………8 Figure 4. Cell rupture methods and their classification……….………13 Figure 5. Antimicrobial activity against S. aureus determined ………..………..24 by the agar well diffusion method

Figure 6. Mean inhibition zones produced by non-lysed (NL), ………..……….25 ultrasonicated (US) and freeze-thawed ultrasonicated (FT+US)

L. acidophilus treatments against bacterial test organisms

Figure 7. L. plantarum 299v antimicrobial activity against C. acnes ………….….…26 determined by the agar well diffusion method

Figure 8. Mean inhibition zones produced by non-lysed (NL), ……….……….27 ultrasonicated (US) and freeze-thawed ultrasonicated (FT+US)

L. plantarum 299v treatments against C. acnes

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List of Tables

Table 1. Bioactives from probiotics that maintain skin barrier integrity………..9 Table 2. Comparison of Lactobacillus viability after sonication and ………..21 freeze-thawing

Table 3. Total protein concentration of two Lactobacillus treatments…………..….22 Table 4. Supernatant pH values used for the agar well diffusion method…………28

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Contents

Abstract……….V Abbreviations……….VII List of figures……….VIII List of tables………IX

Chapter 1 – Introduction………..………1

Chapter 2 – Literature review……….………...3

2.1 Skin structure and microbiota………...………3

2.2 Probiotics and postbiotics for skin………7

2.2.1 Current postbiotic studies……….…...9

2.2.2 Postbiotic use advantages……….….10

2.2.3 Lactobacillus acidophilus and Lactobacillus plantarum as …………...11

Postbiotics 2.2.4 Current state of cosmetic products containing postbiotics……….12

2.3 Biotechnological processes for lysates/postbiotics obtention………….………..12

2.3.1 Mechanical methods: ultrasonication……….….……..13

2.3.2 Physical methods: thermal treatments………..14

Chapter 3 - Hypothesis, general and specific objectives……….………15

3.1 Hypothesis………15

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3.2 General objective……….…15

3.3 Specific objectives………...15

Chapter 4 - Materials and methods……….16

4.1 Inoculum activation of probiotics and pathogenic bacteria………...16

4.2 Probiotic cellular lysis through ultrasonication………17

4.2.1 Plate count for disruption confirmation……….18

4.3 Total protein quantification……… 18

4.4 Antimicrobial activity………...18

Chapter 5 - Results and discussion………...20

5.1 Cellular disruption………20

5.2 Total protein quantification……….………21

5.3 Antimicrobial activity………...23

Chapter 6 - Conclusions and future work……….29

6.1 Conclusions………..29

6.2 Future work………-………..30

Chapter 7 – Bibliography………...……...31

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Chapter 1 Introduction

Skin diseases affect the quality of life of patients because of the social rejection and the emotional impact associated to them. External and internal factors such as climate, hygiene, diet and hormonal changes play specific roles on skin pathologies, however commensal bacteria are key players of immunological responses and skin defense mechanisms (Yu et. al, 2020). Commensal bacteria are part of a complex community of microorganisms called the skin microbiota (Mottin and Suyenaga, 2018). The composition of this bacterial communities depends on the skin characteristics that are generated by different temperature, pH, moisture and sebum content across follicles. Imbalances in microbial communities allow the growth and colonization of opportunistic species, resulting in disease (Traisaeng et. al, 2019).

Acne vulgaris, atopic dermatitis and other skin pathologies associated to overgrowth of opportunistic bacteria require intensive treatment use and have a profound emotional impact (Goodarzi et. al, 2020). Currently, there’s no cure for these diseases and there’s so much more to elucidate regarding their origin and the role of the skin microbiome (Mottin and Suyenaga, 2018). Conventional treatments include the prolonged use of antibiotics or retinoic acids, and topical corticosteroids for acne and atopic dermatitis, respectively (Sadeghzadeh-Bazargan et. al, 2020).

However, long-term use of this actives has shown adverse effects that go from mild to severe. For acne vulgaris, antibiotic resistance, systemic inflammation and alteration of the skin microbiota side effects have been documented. On the other hand, corticosteroid application results in skin thinning, rosacea and melanocyte inhibition together with a loss of the treatment benefits through time (Atherton, 2003;

Coondoo et. al, 2014).

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Restoration of the skin microbiome functions such as topically applying probiotic and postbiotic-derived bioactives has shown positive results for ameliorating this skin diseases while decreasing adverse effects of conventional treatment use (Nole, Yim and Keri, 2014).

For this work, probiotic lysates from L. acidophilus and L. plantarum 299v were evaluated for their antimicrobial activity against the common skin pathogenic bacteria Cutibacterium acnes, Streptococcus pyogenes and Staphylococcus aureus.

Results show an antimicrobial activity of L. acidophilus lysates against the 3 studied pathogens, while L. plantarum 299v lysates had a specific antimicrobial effect against C. acnes.

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Chapter 2

Literature review

2.1 Skin structure

The skin is the largest organ in the body and the first defense barrier against external factors such as radiation, chemicals and pathogens. It has an important role in fluid exchange through sweat, regulation of the body’s temperature to achieve homeostasis and as a mechanical barrier for injuries (Yousef and Sharma, 2019). It consists of two layers: epidermis and dermis. Some authors include a third layer called the hypodermis or subcutaneous fascia because of its proximity with the other two layers (Barcaui et. al, 2015). Each layer consists of different cellular types and inner layers, being the epidermis the thickest and outermost layer of the skin (Boer et. al, 2016).

The stratum corneum is the outermost layer of the epidermis, consisting of corneocytes held by lipids such as cholesterol, fatty acids and ceramides arranged in a brick-and-mortar model, called the protective skin barrier (Salem et. al, 2018).

This barrier maintains correct water levels on the skin, retains skin moisture and prevents the colonization or invasion of pathogenic microorganisms (Boer et al, 2016). Together with sweat glands such as eccrine glands, which secrete sweat with salts, the skin barrier is slightly acidified to a pH of 4.6 to 5.6, creating an acid mantle that also has a protective function (Schmid-Wendtner, 2005).

A major epidermis component is a collective set of microorganisms called the cutaneous microbiota, which consists of commensal and mutualistic microbial communities such as bacteria, archaea, fungi, viruses and parasites that inhabit the human skin throughout the body (Belkaid and Segre, 2016; Chen, Fischbach and Belkaid, 2018). Their distribution depends on the different microenvironments present in the skin that vary on pH, moisture content and topography, creating the

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environmental conditions for these skin microbes to proliferate and survive (Rosenthal et. al, 2011; Grice, 2014; Sanford and Gallo, 2014).

From all the microorganisms that conform the skin microbiome, bacterial strains have a profound impact and more complex interactions with the human skin.

There’s an important equilibrium and protection relationship among these microorganisms and the skin that is achieved through antimicrobial peptide production, immunomodulation, inhibition of pathogen growth through niche competition and maintenance of epidermal integrity through keratinocyte interactions (Lukic et. al, 2017; Goodarzi et. al, 2020).

Figure 1. Comparison between a healthy skin barrier versus a damaged skin barrier.

Different niches are created due to the variety of environments that allow bacterial growth. In sebaceous areas like the face, Cutibacterium and Staphylococcus species can be vastly found, while Corynebacterium dominates in moist areas. As for dry sites, β-Proteobacteria and Flavobacteriale are more likely to be found (Prescott et. al, 2017). The skin microbiome is shaped since birth to

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adulthood, shifting greatly during puberty with increasing abundance of Corynebacterium and Cutibacterium species. Overall, the dominant phyla are Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, while the dominant genra are Staphylococcus, Cutibacterium, Corynebacterium and Streptococcus (Rosenthal et. al, 2011; Grice, 2014; Belkaid and Segre, 2016).

Changes such as alterations derived from external factors, can alter the immune response and modify the skin microbiome composition, resulting in an imbalance of the microbial communities (Figure 1). This skin dysbiosis triggers the development of non-infectious pathologies like acne, atopic dermatitis and psoriasis (Lee et. al, 2018; Byrd, Belkaid and Segre, 2018).

There are three common pathogens that can behave as opportunistic bacteria, rather than commensal strains and cause some of these pathologies. Acne vulgaris a disease caused by an imbalance of Cutibacterium acnes (formerly known as Propionibacterium acnes) populations, a bacterium that proliferates in lipophilic and anaerobic environments like the sebaceous glands, representing 20-70%

predominance on these sites (Beylot et. al, 2013). Interactions among the host, the environment and the microbiome cause lesions and metabolomic shifts that initiate acne (Figure 2). The severe acne cases have been shown to be caused by C. acnes biofilm formation and production of mixed metabolites that promote persistent inflammation. The main factors that contribute to this dysbiosis are androgen- mediated seborrhea and a lack of regulation if sebum production. Interestingly, C.

acnes is a regular skin commensal that also has protective effects on the skin, for example against Staphylococcus epidermidis or Staphylococcus. aureus, by follicle colonization and secretion of bacteriocins and fatty acids that create inhospitable environments (Xu and Li, 2019)

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Depending on the strain species and its phylotype, they can either inhibit other bacteria proliferation or overpopulate the skin microenvironments causing disease.

In the case of S. epidermidis, it can decrease C. acnes growth through production of succinic acid and antibacterial polymorphic toxins (O’Neill and Gallo, 2018).

Altogether, both C. acnes and S. epidermidis have important roles in acne modulation and development. Further research is still needed to elucidate the molecular mechanisms that shift bacterial commensalism to disease.

Figure 2. Acne vulgaris biogenesis and progression. (Adapted from Qidwai et. al, 2017)

Atopic dermatitis is a chronic inflammatory skin disease that is generated by alterations in the immune response because of environmental factors or genetics that contribute to an impaired skin barrier (Atherton, 2003). The main genetic alteration is loss of functionality in the gene encoding filaggrin, a water retaining and epidermal homeostasis protein. Higher pathogenic invasion and colonization is also a factor that worsens atopic dermatitis that is caused by this skin barrier defects (Salem et. al, 2018).

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Staphylococcus aureus is abundant within atopic dermatitis patients with decreased microbial diversity that is also associated with disease severity.

Therefore, replenishing the skin microbial communities has been associated with controlling S. aureus growth and regaining microbiome diversification (Yu. et. al, 2020).

An infrequent but usually skin pathogenic microorganism is Streptococcus pyogenes, the “flesh-eating” bacteria. It has the potential to adhere to the surface of the skin and depending on the tissue where the infection occurs, it can cause mild to severe harm. S. pyogenes infections include impetigo as the most common disease. Deeper skin lesions include connective tissue and adipocyte infections like cellulitis, erysipelas and necrotizing fasciitis (Cogen, Nizen and Gallo, 2009).

Probiotics and postbiotics have been proved as useful agents to prevent and treat cutaneous diseases such as acne and atopic dermatitis through modulation and restoration of skin microbial balance. Mainly, administration of oral probiotics such as Lactobacillus and Bifidobacterium spp. have been studied as possible treatments. Recently, topical administration of this actives in cream or lotion preparations has been explored for protection against S. aureus in patients with atopic dermatitis. Gut commensal bacteria use, such as Lactobacillus johnsonii and Streptococcus thermophilus, have shown favorable results after a 2- and 3-week topical application study atopic dermatitis patients (Yu et. al, 2020; Mottin and Suyenaga, 2018)

2.2 Probiotics and postbiotics for skin

Among the skin’s commensal bacteria, there are some that exert beneficial effects in maintaining skin health. These bacteria are called probiotics and are defined as

“live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002; Sarao and Arora, 2017) The first studies about probiotics involved its effects in the gastrointestinal tract and received the name of “gut microflora” (Dixit, Wagle and Vakil, 2016). In vitro studies using probiotic strains such as Lactobacillus rhamnosus and Lactobacillus reuteri, have

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shown potential cellular components or metabolites that can protect keratinocytes from S. aureus infection (Prince, McBain and O’Neill, 2012). The protective effects are thought to be caused by exclusion of pathogen colonization, inhibition of pathogen growth and their displacement from keratinocytes. Besides, it is known that skin health maintenance is strain dependent because of the release of specific beneficial metabolites (Mohammedsaeed et. al, 2014).

Figure 3. Probiotic and postbiotic characteristics with examples.

A relatively new concept in the probiotic field is postbiotics. Postbiotics are soluble factors and metabolic by-products that are secreted in the cell media or are obtained through cellular rupture (Figure 3). Examples of postbiotics include organic acids, peptidoglycans, bacteriocins, fatty acids, hyaluronic acid and intracellular enzymes (Table 1) (Aguilar-Toalá et. al, 2018)

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Table 1. Bioactives from probiotics that maintain skin barrier integrity.

Bioactive Effect Tissue activity Reference

Organic acids

Antimicrobial activity Maintenance of the acid

mantle

Promote moisturization

Epidermis and dermis

Lew and Liong, 2013 Cinque et. al,

2011 Diacetyl Antimicrobial activity against

gram-negative bacteria Stratum corneum Lew and Liong, 2013

Fatty acids

Prevention of TEWL Compose the lipid mantle

Stimulation of host cells Antimicrobial activity

Stratum corneum

Lew and Liong, 2013 Cinque et. al,

2011 Chen et. al,

2018 Sphingomyelinase

Ceramide production through sphingomyelin

hydrolyzation Immunomodulatory effects

Stratum corneum

Lew and Liong, 2013 Chen et. al,

2018 Hyaluronic acid Increase skin hydration

Improve elasticity

Stratum granulosum, Stratum spinosum, Stratum basale and

dermis

Lew and Liong, 2013 Cinque et. al,

2011 Bacteriocins Antimicrobial activity against

C. acnes

Regulation of inflammation Epidermis and dermis

Oh et. al, 2006 Lew and Liong, 2013 Peptidoglycans Stimulation of human β-

defensins and cathelicidins Dermis Lew and Liong, 2013

2.2.1 Current postbiotic studies

Lysates and soluble metabolites of L. acidophilus NCFM®, Bifidobacterium lactis 420, L. acidophilus La-14, L. salivarius Ls-33 and Propionibacterium jensenii P63, were added to keratinocytes, demonstrating that soluble metabolites increased the expression of claudin-4. L. acidophilus NCFM lysates increased the expression of zonula occludens-1 and occludin, which are tight junction proteins. These effects

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were observed in a species-dependent effect and help to restore the tight junctions between keratinocytes, improving the skin barrier function (Putaala et. al, 2012)

Another study involved the obtention of bacteriocins from Lactococcus spp.

HY449 cell free supernatant, resulting in inhibition of the growth of common skin inflammatory bacteria such as S. epidermidis, S. aureus and S. pyogenes.

Bacteriocin recollection occurred through filtration and purification of the spent culture media and resulted in a useful antimicrobial component that can also inactivate C. acnes (Oh et. al, 2006).

Lactobacillus plantarum extracts could be useful in reduction of growth of microbial populations through stimulation of beta-defensins production in skin cells (Sullivan et. al, 2005). These extracts include the use of a heat exchange-treated extract, cell disruption and a heat exchange-treated extract subjected through cross- flow filtration, containing a filtrate and retenate. All the extracts showed defensin- inducing activity that could be useful for treating acne because of the reduction effect in inflamed and non-inflamed acne lesions when used in a 2-month period (Cinque et. al, 2011).

Using Aloe Vera fermented supernatant by L. plantarum HM218749.1, scavenging capacities were observed as well as inhibition zones for several pathogens including C. acnes. The highest inhibition zone observed was against C.

acnes and consisted of 27 mm. Anti-inflammatory effects of this supernatant were also evaluated, demonstrating a reduction in production of interleukin-1, Interleukin- 6 and Tumor Necrosis Factor-⍺ at mRNA and protein levels. The authors suggest that this ingredient can be used in cosmetic formulations to treat diseases because of its anti-inflammatory and antimicrobial potential (Jiang et. al, 2016).

2.2.2 Postbiotic use advantages

After probiotics, postbiotics are a preferred ingredient in topical formulations because of their less complex handling. Probiotic use possesses a challenge in maintaining the viability of live bacteria, as well as higher complexity in

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manufacturing and storage of the final cosmetic product (Huang and Tang, 2015).

When compared to probiotics, postbiotics offer another advantage, which is obtention of higher bioactive concentrations such as the case of lysates due to bacterial disruption. Besides lysates, studies in the postbiotic field have been done using fermentation products like as proteins, filtrates and supernatants (Zgoła- Grześkowiak et. al, 2016).

2.2.3 Lactobacillus plantarum and Lactobacillus acidophilus as postbiotics Lactic acid bacteria have been widely used for production of dairy products, oral supplements and functional foods to restore the gut microbiota because of their potential to inhibit pathogenic growth (Bhola and Bhadekar, 2019). L acidophilus is the most important lactobacilli because of its capacity to secrete antimicrobial substances with activity over different pH conditions such as bacteriocins and other peptides (Saad, 2015). In a recent study by Tayupanta et. al, 2018, L. acidophilus has been studied as a probiotic strain with antimicrobial activity against C. acnes.

Lactobacillus plantarum is also an important probiotic that can be found in the environment and foods. It is also present in the human body, in places like the oral cavity, intestinal tract and feces (Jiang et. al, 2016) The main characteristics of L.

plantarum are prevention of inflammatory responses through anti-inflammatory cytokine production and adhesion to epithelial cells by collagen and fibronectin interactions (Beck et. al, 2009; Melgar-Lalanne and Hernández, 2012) making it an attractive probiotic for skin related applications.

Both probiotic bacteria have shown to be metabolically flexible, with broad applications and adaptations to different environments. The main characteristics that make them an interesting active to be used in formulations for improving skin diseases are is safe utilization, extensive research and acid tolerability, indicating its possible survival in the skin acid mantle (Liu et al., 2009; Wren Laboratories, 2020).

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2.2.4 Current state of cosmetic products containing postbiotics

Current cosmetic products containing probiotic and postbiotic ingredients include serums, face masks, moisturizers, cleansers and creams mainly containing live lactic acid bacteria, Lactobacillus ferments, Lactobacillus ferment lysates, Bifida ferment lysates and yogurt. This skincare products are targeted to restore the skin’s protective barrier, increase skin luminosity, reduce redness through soothing effects and decrease fine lines and wrinkles, according to the claims in available cosmetic products in the American, European and Korean market.

Regardless, the cosmetic preservation field in the use of live bacteria is still complex because normally the addition of preservatives is directed towards the inhibition of microbial growth by using broad-spectrum antimicrobials or combination of several preservatives in cosmetic products with high water content (Zgoła- Grześkowiak et. al, 2016). Therefore, bacterial viability at long term, as well as storage conditions and selection of the preservative ingredient that allows probiotic bacteria growth, are still a big challenge. This is another reason why postbiotics are more used and explored than probiotics.

Still, all the underlying interactions between the skin, immune cells and the microbiota are largely unknown, therefore more exploration is needed in this field to understand the exact mechanisms that aid in skin repair and homeostasis recovery.

Also, since the skin modulatory effects are strain dependent among the probiotic, its metabolites and the response of the pathogenic bacteria, performing in vitro studies with L. acidophilus and L. plantarum 299v could provide a solid scientific foundation behind the use of postbiotics in cosmetic products.

2.3 Biotechnological processes for lysates/postbiotics obtention

Cellular rupture is a commonly used technique in biotechnological process because it allows the recovery of intracellular products and its classified in mechanical and non-mechanical methods. Mechanical rupture includes bead milling, high-pressure homogenization and ultrasonication, two methods commonly used at

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large scale because of the ease of scalability and application to lyse different cell types. Non-mechanical methods are divided into physical, chemical and enzymatic or biological processes (Figure 4) that include the use of solvents, chaotropic agents and enzymes that aid in rupture of the cellular membrane and destabilization of osmotic pressures. In bacteria, the cell membrane structure allows the maintenance of intracellular metabolism and transport of molecules (Harrison, 2011)

2.3.1 Mechanical methods: ultrasonication

Ultrasonication uses high frequency sound to generate liquid cavitation. This phenomenon occurs by the propagation of ultrasound in the liquid through oscillations, forming vapor bubbles that grow until maximum expansion, resulting in a violent collapse (Huang et. al, 2017). The acoustic range used is from 20 to 250 W with minimal frequencies of 20 kHz. Different sample volumes, wave amplitude frequencies and time are selected depending on the ultrasonication objective and the bacterial strain. Higher intensities cause more cellular rupture in contrast with an increase in volume (Mota et. al, 2018).

Figure 4. Cell rupture methods and their classification (from Harrison, 2011).

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2.3.2 Physical methods: thermal treatments

Thermal treatments can cause cell rupture by the application of elevated temperatures or freezing and thawing cycles. Both processes result in breakage of the cell membrane through protein denaturalization or formation of ice crystals (Shehadul Islam, Aryasomayajula and Selvaganapathy, 2017). Nonetheless, thermal processes are not always applicable for intracellular product obtention since protein denaturation, inactivation or precipitation can occur (Haddaji et. al, 2015).

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Chapter 3

General and specific objectives

3.1 Hypothesis

Postbiotic solutions obtained from and Lactobacillus acidophilus and Lactobacillus plantarum 299v could have antimicrobial activity against Cutibacterium acnes, Streptococcus pyogenes and Staphylococcus aureus.

3.2 General objective

The objective of this study is to evaluate the postbiotic antimicrobial effects of L. acidophilus and L. plantarum 299v ultrasonicated, freeze-thawed ultrasonicated lysates, heat treated cells and supernatants against Cutibacterium acnes, Streptococcus pyogenes and Staphylococcus aureus.

3.3 Specific objectives

I. Determine the effect of ultrasonication and heat treatments to produce postbiotic solutions through cellular viability evaluation and quantification of lysates total protein of probiotics spp.

II. Assessment of antimicrobial activity of lysates and supernatants using the agar well diffusion method.

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Chapter 4

Materials and methods

4.1 Inoculum activation of probiotics and pathogenic bacteria

L. acidophilus (ATCC 4356) and L. plantarum 299v (DSM 9843) were obtained from a previously prepared and frozen stock (Nutriomics group collection).

For activation, cells were inoculated in BD Difco™ Lactobacilli MRS broth (Fisher Scientific, USA) and were incubated at 37°C for 20 hrs (1535 Incubator, Shel Lab).

After incubation, bacteria inoculums were transferred into new MRS broth to produce biomass at the same incubation conditions for 20 h. Cells were harvested by centrifugation at 10,000 x g for 5 min at 20°C (SL16R centrifuge, Thermo Scientific) and washed three times with 25 ml of PBS 1x (Phosphate 0.1M, NaCl 0.15M, pH 7.2) using vortex homogenization of 15-30 s at maximum speed between washes.

The supernatant was decanted and stored at 4°C for posterior use. The pellet was weighed to 0.68 g. PBS 1x was added and the samples were adjusted to a final concentration of 1.7% w/v (~1.45x109 CFU/ml). From the four samples, two tubes for each bacterium were stored at -20°C for the freeze thawing + ultrasonication treatment (FT+US) and the remaining two samples for posterior ultrasonication (US) were stored at 4°C until use.

S. aureus (ATCC 25923) and S. pyogenes (ATCC 19615) were grown in commercial BHI broth (BD BBL™ Brain Heart Infusion) and incubated at 37°C for 18 hours on aerobic conditions. The inoculum was centrifuged at 10,000 x g for 10 min at 20°C. Supernatant was discarded and the remaining pellet was washed twice 10 ml of PBS 1x and homogenized through vortexing for 30 s. The resulting pellet was homogenized and transferred to 190 ml of fresh BHI broth for posterior incubation at 37°C for 18 hours. To prepare the stock tubes, bacterial cultures were centrifuged at 10,000 x g for 10 min at 20°C and washed three times with 25 ml of PBS 1x. After

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washing, the pellet was homogenized with 12 ml of PBS 1x and 3 ml glycerol (20%).

The final sample was distributed into 2 ml tubes and stored at -80°C.

From a C. acnes (ATCC 11827) stock, 100 µl were added to 9 ml of Reinforced Clostridial broth (BD Difco™ Reinforced Clostridial Medium) and incubated at 37°C for 48 h in an anaerobiosis jar (Oxoid Anaero Jar™, Thermofisher Scientific) with a gas pack container system (GasPack™ Ez, BD). The inoculum was centrifuged 2 times at 10,000 x g for 10 min at 25°C and the supernatant was removed. The pellet was resuspended in 5 ml of fresh Reinforced Clostridial broth and transferred to 22.5 ml of the same broth. Incubation was done at 37°C for 48 h in anaerobiosis. After incubation, the culture was centrifuged again at 10,000 x g for 10 min at 25°C. The pellet was resuspended with 100 ml of Reinforced Clostridial broth and added to a jar with 100 ml of the same broth for incubation at 37°C for 48 h. Samples were centrifuged at 10,000 x g for 10 min at 25°C and washed once with PBS 1x. Prior to freezing, the pellet was transferred to 12 ml of Reinforced Clostridial broth and 3 ml of glycerol (20%) were added. Samples were stored at -80°C until use.

4.2 Probiotic cellular lysis through ultrasonication

Prior to sonication, FT+US samples from L. acidophilus and L. plantarum 299v at a 1.7% w/v concentration, were thawed at 25°C. Both US and FT+US were vortexed and then sonicated using (Q125 sonicator, QSonica) for 10 cycles of 60 s pulse-on with 20 s pulse-off using an amplitude of 70% and a frequency of 125 kHz.

The probiotic samples (US and FT+US) were cooled by placing them on ice during the sonication process. To avoid contamination, the probe was sanitized with 70%

ethanol and followed by a sterile PBS 1x wash for 1 min.

For the thermal treatment, after sonication, an aliquot of each probiotic was placed on a water bath for 1.5 h at 80°C (Water Bath model 1217, VWR). This step was applied in order to evaluate the effect of heat-induced denaturalization on bacteriocins, which are peptides with antimicrobial activity.

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4.2.1 Plate count for disruption confirmation

Evaluation of cellular rupture was done using the serial dilutions and plating method, adding 100 µl of the US and FT+US sonicated treatments from each probiotic bacteria diluted with sterile PBS 1x for the 101 and 102 dilutions. To plate, 100 µl of the three dilutions were added to a sterile commercial MRS agar (BD Difco™ Lactobacilli MRS agar) plate and hand plated (Drigalski-spatula technique).

Plates were then incubated at 37°C for 20 h and CFU/ml were compared.

4.3 Total protein quantification

Total protein quantification was done using the Pierce™ BCA Protein Assay Kit (Catalog No. 23225, Thermofisher) according to the instructions of the manufacturer. To calculate the working reagent volume for the microplate procedure, Equation 1 was used. The number of standards correspond to the calibration curve (Bovine Serum Albumin, 2 mg/ml) of the diluted BSA standards and the number of unknowns to the samples (NL, US, FT+US, HT, HT-US and HT-FT+US). The final volume of reagent A was 12 ml and of reagent B (50 parts of A) was 240 µl, giving a final working reagent volume of 12.240 ml

Volume working reagent = (# standards + # unknowns) x (# replicates) x (200 µl) (Eq. 1)

For each microplate well, 25 µl of the sample were added to each well, followed by 200 µl of the working reagent. The microplate was incubated at 37°C for 30 min.

After incubation, the plate was set to 25°C and the absorbance was read at 562 nm (Synergy HT microplate reader, Biotek).

4.4 Antimicrobial activity

From the previously prepared stocks of each pathogenic bacteria, 100 µl of C. acnes and S. pyogenes were added to 10 ml of Reinforced Clostridial and BHI broth respectively. The inoculums were incubated at 37°C for 48 h under anaerobic conditions. For S. aureus, 100 µl were grown in Mueller-Hinton broth and incubated

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at 37°C for 20 hr under aerobic conditions. Then, using an inoculation loop, each strain was seeded into Differential Reinforced Clostridial, BHI or Mueller-Hinton agar plates and incubated at the same conditions to obtain single colonies.

Five isolated single colonies were added to 5 ml of fresh broth and incubated until cellular suspensions reached an optical density of 0.08-1 at 625 nm (Genesys 10S UV,-Vis Spectrophotometer, Thermofisher Scientific) according to the methodology of (Cona, 2002) as an equivalent to a 0.5 McFarland turbidity standard.

Cell suspensions were then inoculated into agar plates using a sterile swab as follows: C. acnes was seeded into Differential Reinforced Clostridial agar, S.

pyogenes in BHI and S. aureus in Mueller-Hinton. Using 200 µl sterile micropipette tips, 6 mm wells were cut by pressing the tips in the pre-poured inoculated agar plates. The probiotic supernatants were used with to two different pH values, final growth and neutral, to evaluate if the inhibitory effects were not attributed to media acidity. Two aliquots from each lactobacilo supernatant were used directly, while the other aliquot was adjusted to neutral pH. After adjustments, the four supernatants were filtered with 0.22 µm sterile syringe filters to remove any remaining bacteria.

For each treatment (non-lysed, US, FT+US, non-lysed HT, HT-US, HT- FT+US and supernatants), 100 µl were added to each well and tested for formation of inhibition zones after incubation at 37°C for 20 h. Antibiotic disks of ceftriaxone and doxycycline were used as positive controls and PBS 1x as negative control. The experiment was done in triplicate. Resulting inhibition zones were measured with a ruler.

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Chapter 5

Results and Discussion

5.1 Cellular disruption

Lactobacillus spp. US and FT+US viability after sonication was compared to non-lysed cells used as control. This assay was done to evaluate the most efficient sonication treatment and the combination of two disruption methods such as freeze- thawing and ultrasonication on the 1.7% w/v samples. Statistical analysis (Table 2), show that the only significant treatment that decreases bacterial viability is FT+US for both lactobacilli. It has been reported that the ultrasonic phenomena can increase or decrease bacterial viability depending on the strain and the amplitude of the ultrasonic wave used. Hor et. al (2014) analyzed the effect of different amplitude waves (20, 60 and 100%) on Lactobacillus spp. strains, including L. acidophilus FTDC 1231. Although viability was not significant, at a 60% amplitude and t= 1 min, there was a log CFU/ml of 7.22 ± 0.09, which indicated almost the same viability compared to the non-lysed control of this study (7.21 ± 0.14). For 100% wave amplitude and t= 3 min, the log CFU/ml value was of 7.23 ± 0.09, demonstrating a null ultrasonication effect. On the other hand, L. casei FTDC 8313 and L. gasseri FTDC 8131 showed a significant viability increase with log CFU/ml values of 6.98 ± 0.12 to 7.35 ± 0.2. A similar effect was observed by Gholamhosseinpour and Hashemi (2018), where L. plantarum AF1 fermented milk received an ultrasound pretreatment at different times (5, 10 and 15 min) and resulted in increased bacterial counts. The higher viability effect was reported at 10 and 15 min of ultrasonication with a 9 - 10 log CFU/ml count. The increase in bacterial viability has been explained as a consequence of an improved cellular permeability due to pore transference of metabolites such as intracellular enzymes or peptides that enhance cellular growth and repair (Wu et al., 2000; Mota et. al, 2018).Also, separation of bacterial clusters caused by the ultrasonication process can increase bacterial viability (Hor et. al, 2014).

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Table 2. Comparison of Lactobacillus viability after sonication and freeze-thawing.

Treatments Viable counts (log CFU/ml)

L. acidophilus L. plantarum 299v

Non-lysed (C) 9.29 ± 0.04 9.16 ± 0.04

US 9.30 ± 0.06 9.14 ± 0.02

FT+US 8.78 ± 0.04 * 8.90 ± 0.03 *

Non-lysed cells were used as control (C). US: Ultrasonicated cells. FT+US:

Freeze-thawed and ultrasonicated cells.

The logarithm of CFU/ml is expressed (mean ± SD). Plate counting was done using triplicates (n=3) and statistical analysis was done using one-way ANOVA and the Tukey test.

* Represents statistically significant results (p<0.05)

The only significant treatment was the combination of freeze-thawing and ultrasonication. Murga, de Valdez and Disalvo (2000) evaluated the viability of Lactobacillus acidophilus colonies after freezing and thawing cycles, through the calculation of zeta potential, observing a lower count when compared to cultures with addition of cryoprotective agents. The authors concluded that the decrease in viability was generated by cell surface injuries. In a study conducted by Wang et. al (2020), L. plantarum subjected to freeze-thawing indicated that the decrease in viability was also caused by changes in the membrane, affecting integrity, permeability and enzymatic activity. However, these effects do not occur in all lactobacilli strains since sensitivity changes depending on the probiotic bacteria.

5.2 Total protein quantification

Protein quantification show that there’s higher protein concentrations in the L.

acidophilus US treatment compared to non-lysed and FT+US treatments. For L.

plantarum 299v, both US and FT+US showed a higher protein concentration compared to non-lysed cells (Table 3). Freezing and thawing aids in peptide release caused by membrane rupture due to ice crystals formation, therefore, higher protein quantification values are obtained compared to non-lysed cells (Wang et. al, 2020).

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Table 3. Total protein concentration of two Lactobacillus treatments.

Treatment L. acidophilus

(µg/ml) L. plantarum 299v (µg/ml)

Non-lysed 2.57 ± 0.10 * 3.64 ± 0.16 *

US 4.03 ± 0.87 3.88 ± 0.65

FT+US 3.33 ± 0.67 3.92 ± 0.56

HT 4.42 ± 0.49 * 5.68 ± 0.64 *

HT-US 4.62 ± 0.10 * 5.13 ± 0.25

HT-FT+US 4.80 ± 0.64 * 5.87 ± 0.14 *

Non-lysed cells were used as control (C). US: Ultrasonicated cells. FT+US:

Freeze-thawed and ultrasonicated cells.

Comparison of total protein concentrations (mean ± SD). Quantification was done using triplicates (n=3) and statistical analysis were evaluated using one-way ANOVA and the Tukey test.

* Represents statistically significant results (p<0.05).

The highest protein concentrations reported correspond to the treatments subjected to heat. Thermal lysis using temperatures above 65°C have demonstrated changes in Lactobacillus bulgaricus protein stability, mainly causing denaturation and inactivation of the cell wall and other membrane proteins (Teixeira et. al, 1997).

One of the most notable effects shown by Lee and Kaletunc (2002) indicate that above this temperature, ribosomes and protein denaturation can cause thermal death and disruption of other cellular interactions. Also, temperature effects on protein stability affect protein conformation, causing possible protein unfolding and destabilization through exposure of hydrophobic amino acid residues (van Dijk, Hoogeven and Abeln, 2015). Since the BCA reagent binds to cysteine, tryptophan and tyrosine, it is plausible that changes in the secondary and tertiary structure of bacterial peptides result in higher protein quantification by the binding of the reagent to this available amino acid residues.

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5.3 Antimicrobial activity

Postbiotic solutions as well as non-lysed L. acidophilus cells showed anti- bacterial activity against S. aureus and C. acnes. The largest diameter was observed with the US treatment with a mean value of 16.8 mm (Figure 5b). The only L.

plantarum 299v treatment with an antagonistic effect against S. aureus was the supernatant of pH 3.8 (Figure 5d), while the pathogen S. pyogenes was only susceptible to US and FT+US treatments from L. acidophilus (Figure 6). A study conducted by Bhola and Bhadekar (2019) showed a reduction in inhibition zones when using whole-broth (non-lysed) L. acidophilus bacteria, confirming a direct competitive exclusion effect among the probiotic and the pathogen, as well as metabolite production of acids or bacteriocins. In another study, Hernández-García et. al (2019) described the inhibitory effect of L acidophilus strain SS80 against S.

aureus, with a mean inhibition diameter of 16.7 mm, similar to the 14.0 mm mean inhibition diameter obtained for the non-lysed treatments. Mohammedsaeed et. al (2014) demonstrated that ultrasonicated Lactobacillus lysates (L. rhamnosus and L.

reuteri) have a specific antimicrobial effect against S. aureus, affecting its growth.

Since antibacterial activity has been reported to be caused specifically by live Lactobacillus and not by lysates against S. pyogenes M1 (Khmaladze et. al, 2019), more research is needed to determine the specific metabolites that generate antagonism between ultrasonicated L. acidophilus and S. pyogenes as presented in this study.

Comparison of US and FT+US treatments against S. aureus, does not show a significant difference, indicating that sonication alone could be sufficient to generate inhibition zones against the evaluated pathogens.

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Figure 5. Antimicrobial activity against S. aureus determined by the agar well diffusion method.

a) Doxycycline and ceftriaxone antibiotic disks were used as positive controls. b) L. acidophilus ultrasonicated (US) and freeze-thawed ultrasonicated (FT+US) inhibition zones. c) L.

acidophilus non-lysed and heat-treated (HT) inhibition zones and d) L. plantarum 299v pH 3.8 and pH 7 inhibition zones.

Antimicrobial activity of L. acidophilus is in line with the study conducted by Tayupanta and Ocana (2019), where L. acidophilus ATCC 4356 strain induced inhibition zones against C. acnes in the mean inhibition zone range of 6 – 9.8 mm.

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Variations between inhibition zone diameter values compared to the results obtained in this study could be due to the use a different C. acnes strain (Lopes et. al, 2017)

Figure 6. Mean inhibition zones produced by non-lysed, ultrasonicated and freeze- thawed ultrasonicated L. acidophilus treatments against bacterial test organisms.

Non-lysed cells were used as control. US: Ultrasonicated cells. FT+US: Freeze-thawed and ultrasonicated cells.

Statistical analysis was done using one-way ANOVA and the Tukey test.

* Represents statistically significant results (p<0.05).

C. acnes was the most sensitive microorganism to L. plantarum 299v treatments (Figure 7), forming inhibition zones with non-lysed, US, FT+US and the supernatant with pH 3.8. The largest diameter was also observed with the US treatment with a mean value of 19 mm (Figure 8). The non-lysed cells inhibitory effect is similar to the results reported by Al-Ghazzewi and Tester (2010), where different Lactobacillus spp. strains, including L. plantarum, showed inhibition against C. acnes strain NCTC 737 growth with a mean diameter range of 10-15 mm.

14.0

16.8 15.0

13.7

16.3 13.3 13.3

11.3

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

NL US FT+US

Mean inhibition zone (mm)

Treatment

S. aureus C. acnes S. pyogenes

*

*

*

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Figure 7. L. plantarum 299v antimicrobial activity against C. acnes determined by the agar well diffusion method.

a) Doxycycline and ceftriaxone antibiotic disks were used as positive controls. b) L. plantarum 299v ultrasonicated (US) and freeze-thawed ultrasonicated (FT+US) inhibition zones and c) L. plantarum 299v non-lysed and heat treated (HT) inhibition zones

Neither L. acidophilus nor L. plantarum 299v HT, HT-US and HT-FT+US treatments formed inhibition zones against the three pathogenic strains evaluated in the study. This result is confirmed by the Mohammedsaeed et. al (2014) study, were antimicrobial activity of the lysates was completely lost when subjecting them to a heat treatment of 100°C for 10 min.

From the evaluated supernatants from both probiotic cultures, only L.

plantarum 299v non-neutralized pH showed antibacterial activity against S. aureus and C. acnes (Figure 6 and Figure 7). According to Arena et. al (2016), the neutralized supernatant of 40 L. plantarum strains evaluated as a control strategy versus Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7 and Staphylococcus aureus, did not show any antimicrobial activity. Analysis by Tejero-Sariñena et. al (2012) demonstrated that probiotic antimicrobial activity is directly correlated to the presence of organic acids in the supernatants generated from glucose, a carbohydrate source in the MRS broth. Hence, identification and quantification of this acids can explain the observed antimicrobial activity effect.

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Figure 8. Mean inhibition zones produced by non-lysed, ultrasonicated and freeze- thawed ultrasonicated L. plantarum 299v treatments against C. acnes.

Non-lysed cells were used as control (C). US: Ultrasonicated cells. FT+US:

Freeze-thawed and ultrasonicated cells.

Wells were evaluated in triplicates (n=3) and statistical analysis was done using one-way ANOVA and the Tukey test.

* Represents statistically significant results (p<0.05).

Since the pH value was lower in L. plantarum 299v supernatant compared to L. acidophilus (Table 4), a higher acid concentration is directly correlated to formation of greater inhibition zones. Lactic acid is the main organic acid produced in culture media, followed by acetic acid. Other metabolites are hydrogen peroxide and diacetyl that have also antimicrobial nature (Hernández-García, 2019).

However, a study conducted by Koohestan et. al (2018), showed that L. acidophilus cell free supernatant inhibited S. aureus growth with a mean diameter of 15-20 mm, therefore more studies are needed to achieve consistency in the presented results.

10.5 11.5

19

16.5

0 5 10 15 20 25

pH 3.8 NL US FT+US

Inhibition zones (mm)

Treatments C. acnes

*

*

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Table 4. Supernatant pH values used for the agar well diffusion method.

Supernatant pH

L. acidophilus 4.8

L. acidophilus 7.0

L. plantarum 299v 3.8 L. plantarum 299v 7.0

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Chapter 6

Conclusions and Future work

6 .1 Conclusions

Significant protein concentration values using the BCA assay indicate a possible protein, enzymatic and ribosomal denaturation due to a prolonged exposure to heating above 65°C. This effect is confirmed by the complete loss of antimicrobial activity of the 3 heat-treated samples against S. aureus, C. acnes and S. pyogenes.

According to the results obtained in the antimicrobial activity, cellular rupture by combination of a thermal treatment (freeze-thawing) plus a mechanical process (sonication) showed a significant effect on both probiotic strains compared to the non-lysed control, suggesting a decrease in cellular viability caused by damage in the cell membrane. Similarities in non-lysed compared to ultrasonicated cells could be attributed to pipetting and other associated serial dilution errors in the plating process, which affected colony counts, since the highest antimicrobial activities and an increase in protein quantification was observed in ultrasonicated samples.

Antimicrobial activity was observed with the non-lysed, US and FT+US treatments, however this effect is strain-specific among probiotic and pathogenic bacteria interactions.

Altogether, results indicate that postbiotic production through cellular rupture using sonication is a useful alternative to inhibit pathogenic bacteria growth in cutaneous diseases.

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6.2 Future work

In contemplation of the obtained results in this study, exploring different lysis methods such as bead milling or using alternative ultrasonication conditions, could improve cellular rupture efficiencies and an increase in the antagonistic effect of Lactobacilli against the skin pathogenic bacteria evaluated. Besides, the use of mechanical rupture may facilitate the process scalability that could be applied to cosmetic formulation purposes.

Since results also show a strain-specific effect, characterization of specific antimicrobial compounds through chromatographic techniques and evaluation of the anti-inflammatory effects of postbiotic solutions could generate a better understanding on the in vitro interactions of skin commensal bacteria and their application to treat cutaneous diseases.

Changing culture conditions like increasing incubation times can promote a higher antimicrobial metabolite production on both the intracellular environment and the supernatant.

Another approach is testing the postbiotics lysate on complex environments such as 3D skin explants or organ-on-chip systems that can simulate with higher fidelity the skin conditions to elucidate the defense mechanisms behind the probiotic bacteria.

Finally, studying cosmetic formulations and stabilization of specific probiotic derived ingredients through microencapsulation or direct addition in skincare products could be an innovative complement to actual treatments for acne, atopic dermatitis or just for improving the skin barrier capabilities.

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