Optimizing bioreactor conditions for Spirulina fermentation by Lactobacillus helveticus and Kluyveromyces marxianus: Impact on chemical & bioactive properties.

This study focused on optimizing the production of fermented Spirulina (FS) products using a bioactivity-guided strategy with Lactobacillus helveticus B-4526 and Kluyveromyces marxianus Y-329 in a 3-L bioreactor. Various operating conditions, including aeration rates and pH modes, were tested. While both microorganisms thrived under all conditions, the "cascade" mode, controlling dissolved oxygen, enhanced protein hydrolysis and antioxidant activity, as confirmed by SDS-PAGE and DPPH/TEAC assays, respectively. Screening revealed that "cascade" FS significantly decreased viability of colon cancer cells (HT-29) in a dose-dependent manner, with up to a 72 % reduction. Doses ≤ 500 µg mL-1 of "cascade" FS proved safe and effective in suppressing NO release without compromising cellular viability. Additionally, "cascade" FS exhibited diverse volatile organic compounds and reducing the characteristic "seaweed" aroma. These findings highlight "cascade" FS as a promising alternative food source with improved bioactive properties, urging further exploration of its bioactive compounds, particularly bioactive peptides.

Effect of exopolysaccharides yield and addition concentration of Lactobacillus helveticus on the processing characteristics of fermented milk and its mechanism.

Exopolysaccharides (EPS) yield and added concentration of lactic acid bacteria can greatly affect the processing characteristics of fermented milk. In order to investigate the effects and mechanisms of EPS yield and added concentration on fermented milk, researchers extracted EPS from 50 strains of Lactobacillus helvedicus (L. helvedicus) and selected the two strains with the largest difference in EPS yield (L. helvedicus LH18 and L. helvetigus LH33) for subsequent experiments. The physicochemical properties of EPS-LH18 and EPS-LH33 were analyzed. The gel characteristics and protein conformation of fermented milk were studied by means of texture analyzer, rheometer, scanning electron microscopy, nuclear magnetic resonance machine, fluorescence spectrophotometer and circular dichroism. The results indicate that the monosaccharide compositions of EPS-LH18 and EPS-LH33 are the same and have good thermal stability. The texture and rheological properties of L. helveticus LH18 fermented milk are significantly superior to other fermented milk. The reason is that L. helveticus LH18 EPS has the highest yield, which leads to a denser gel structure, lower surface hydrophobicity and free sulfhydryl content of its fermented milk. According to circular dichroism analysis, ß- sheet and random coil are the internal factors leading to the difference in fermented milk gel. In addition, the fermented milk improved even more favorably as the concentration of the two EPS additions increased. As described above, L. helveticus LH18 has the potential to be an excellent yogurt starter, and both of the above EPS can be used as probiotic stabilizer alternatives for fermented dairy products.

Long-term intake of Lactobacillus helveticus enhances bioavailability of omega-3 fatty acids in the mouse retina.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), are required for the structure and function of the retina. Several observational studies indicate that consumption of a diet with relatively high levels of n-3 PUFAs, such as those provided by fish oils, has a protective effect against the development of age-related macular degeneration. Given the accumulating evidence showing the role of gut microbiota in regulating retinal physiology and host lipid metabolism, we evaluated the potential of long-term dietary supplementation with the Gram-positive bacterium Lactobacillus helveticus strain VEL12193 to modulate the retinal n-3 PUFA content. A set of complementary approaches was used to study the impact of such a supplementation on the gut microbiota and host lipid/fatty acid (FA) metabolism. L. helveticus-supplementation was associated with a decrease in retinal saturated FAs (SFAs) and monounsaturated FAs (MUFAs) as well as an increase in retinal n-3 and omega-6 (n-6) PUFAs. Interestingly, supplementation with L. helveticus enriched the retina in C22:5n-3 (docosapentaenoic acid, DPA), C22:6n-3 (DHA), C18:2n-6 (linoleic acid, LA) and C20:3n-6 (dihomo gamma-linolenic acid, DGLA). Long-term consumption of L. helveticus also modulated gut microbiota composition and some changes in OTUs abundance correlated with the retinal FA content. This study provides a proof of concept that targeting the gut microbiota could be an effective strategy to modulate the retinal FA content, including that of protective n-3 PUFAs, thus opening paths for the design of novel preventive and/or therapeutical strategies for retinopathies.

Untargeted metabolomics provide new insights into the implication of Lactobacillus helveticus strains isolated from natural whey starter in methylglyoxal-mediated browning.

Hard cheeses may occasionally show a brown discolouration during ripening due to multifactorial phenomena that involve bacteria and give rise to pyrazines arising from methylglyoxal. The present work aimed at developing a novel approach to investigate the role of natural starters in browning. To this object, 11 strains of L. helveticus were incubated in a medium containing 10 % rennet casein dissolved in whey, and then growth was monitored by measuring pH and number of genomes/mL. Browning was assessed through CIELab analysis, methylglyoxal production was determined by targeted mass spectrometry, and untargeted metabolomics was used to extrapolate marker compounds associated with browning discoloration. The medium allowed the growth of all the strains tested and differences in colour were observed, especially for strain A7 (ΔE* value 15.92 ± 0.27). Noteworthy, this strain was also the higher producer of methylglyoxal (2.44 µg/mL). Metabolomics highlighted pyrazines and ß-carboline compounds as markers of browning at 42 °C and 16 °C, respectively. Moreover, multivariate statistics pointed out differences in free amino acids and oligopeptides linked to proteolysis, while 1,2-propanediol and S-Lactoylglutathione suggested specific detoxification route in methylglyoxal-producing strains. Our model allowed detecting differences in browning amid strains, paving the way towards the study of individual L. helveticus strains to identify the variables leading to discoloration or to study the interaction between different strains in natural whey starters.

The structure of a Lactobacillus helveticus chlorogenic acid esterase and the dynamics of its insertion domain provide insights into substrate binding.

Chlorogenic acid esterases (ChlEs) are a useful class of enzymes that hydrolyze chlorogenic acid (CGA) into caffeic and quinic acids. ChlEs can break down CGA in foods to improve their sensory properties and release caffeic acid in the digestive system to improve the absorption of bioactive compounds. This work presents the structure, molecular dynamics, and biochemical characterization of a ChlE from Lactobacillus helveticus (Lh). Molecular dynamics simulations suggest that substrate access to the active site of LhChlE is modulated by two hairpin loops above the active site. Docking simulations and mutational analysis suggest that two residues within the loops, Gln145 and Lys164 , are important for CGA binding. Lys164 provides a slight substrate preference for CGA, whereas Gln145 is required for efficient turnover. This work is the first to examine the dynamics of a bacterial ChlE and provides insights on substrate binding preference and turnover in this type of enzyme.

Co-cultivation effects of Lactobacillus helveticus SNA12 and Kluveromyces marxiensis GY1 on the probiotic properties, flavor, and digestion in fermented milk.

This study aimed to evaluate inoculating the lactic acid bacteria Lactobacillus helveticus SNA12 and the yeast Kluyveromyces marxiensis GY1 as starter cultures on milk fermentation. In this study, the probiotic properties of L. helveticus SNA12, K. marxiensis GY1 and co-culture of these two strains (L. helveticus SNA12-K. marxiensis GY1) were investigated, and the results showed that K. marxiensis GY1 had better gastrointestinal tolerance, aggregation, and cell adhesion properties than L. helveticus SNA12. After the co-cultivation of two strains, the presence of K. marxiensis GY1 significantly increased the gastrointestinal tolerance, aggregation, and adhesion characteristics of L. helveticus SNA12. In order to investigate the flavor changes, digestive characteristics, and antioxidant properties following co-cultivation fermentation, the optimal fermentation ratio of 8 %-2% (v/v) and fermentation temperature (37 °C) of L. helveticus SNA12-K. marxiensis GY1 were determined. The results of the electronic nose and electronic tongue showed that L. helveticus SNA12-K. marxiensis GY1 could increase the aroma components of fermented milk, such as terpenes and aromatic substances. Meanwhile, dynamic in vitro rat stomach-duodenum model was used to analyse the changes in the digestion of proteins and peptides (<10 kDa), and the results indicated that co-cultivation fermented milk could be digested faster compared to a single fermentation. Furthermore, the antioxidant capacity of co-cultivation fermented milk was higher than that of single fermentation.

Isolation, characterization, and mode of action of a class III bacteriocin produced by Lactobacillus helveticus 34.9.

Traditionally fermented foods and beverages are still produced and consumed at a large scale in Romania. They are rich sources for novel lactic acid bacteria with functional properties and with potential application in food industry or health. Lactobacillus helveticus 34.9, isolated from a home-made fermented milk is able to inhibit the growth of other bacteria, such as other lactic acid bacteria, but also strains of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Halobacillus hunanensis, a halobacterium isolated from the degraded wall of a Romanian monastery. L. helveticus 34.9 produces a large bacteriocin (35 KDa), active in a wide pH range, but inactivated by heat and proteinase K treatment. It shares about 20% sequence coverage with helveticin J, as determined by LC-MS analysis. Bacteriocin production was enhanced under stress conditions, especially when combined stresses were applied. Its mode of action and degree of inhibition depended on the concentration and on the indicator strain that was used; L. delbrueckii subsp. bulgaricus LMG 6901T cells from a suspension were killed, but the viability of H. hunanensis 5Hum cells was only reduced to 60%, within 8 h. However, the bacteriocin was able to prevent the bacterial growth of both indicator strains when added to the cultivation medium prior inoculation. Scanning electron microscopy images revealed morphological changes induced by the bacteriocin treatment in both sensitive strains, but more severe in the case of L. delbrueckii subsp. bulgaricus. Due to the broad antibacterial spectrum and its production under various stress conditions, the bacteriocin or the producing strain may find application in health, food and non-food related fields, including in the restoration of historical buildings.

Co-fermented cow milk protein by Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 attenuates its allergic immune response in Balb/c mice.

Milk protein is one of the major food allergens. As an effective processing method, fermentation may reduce the potential allergenicity of allergens. This study aimed to evaluate the therapeutic potential of co-fermented milk protein using Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 in cow milk protein allergy (CMPA) management. This study determined the secondary and tertiary structures of the fermented versus unfermented proteins by Fourier-transform infrared spectroscopy and surface hydrophobicity to evaluate its conformational changes. Our results showed that different fermentation methods have significantly altered the conformational structures of the cow milk protein, especially the tertiary structure. Further, the potential allergenicity of the fermented cow milk protein was assessed in Balb/c mice, and mice treated with the unfermented milk and phosphate-buffered saline were used as a control. We observed a significant reduction in allergenicity via the results of the spleen index, serum total IgE, specific IgE, histamine, and mouse mast cell protease 1 in the mice treated with the co-fermented milk protein. In addition, we analyzed the cytokines and transcription factors expression levels of spleen and jejunum and confirmed that co-fermentation could effectively reduce the sensitization of cow milk protein by regulating the imbalance of T helper (Th1/Th2 and Treg/Th17). This study suggested that changes of conformational structure could reduce the potential sensitization of cow milk protein; thus, fermentation may be a promising strategy for developing a method of hypoallergenic dairy products.

Release of Leu-Pro-Pro from corn gluten meal by fermentation with a Lactobacillus helveticus strain.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitory peptides are potential alternatives to the synthetic ACE inhibitory drugs, but the in vivo antihypertensive effects of most of them have not been confirmed. The tripeptide Leu-Pro-Pro (LPP) is one of the few peptides that have been proved clinically effective in reducing the blood pressure of hypertensive patients and casein is currently its major source. LPP is contained in multiple fractions of zein, and corn gluten meal (CGM) is hence a potential new source of LPP. For this purpose, CGM was fermented with a Lactobacillus helveticus strain and the medium composition was optimized; the decoloration of the resultant hydrolysate was investigated as well. RESULTS: LPP could be successfully released from CGM by fermentation with the strain Lactobacillus helveticus CICC 22536. The highest LPP content and protein recovery of 561 mg kg-1 and 14.92% occurred in the medium containing 20 g L-1 glucose, 15 g L-1 beef extract, 60 g L-1 CGM, 10 g L-1 CaCO3 , 0.5 g L-1 NaCl, and inoculation amount 6%. The supplementation of Flavourzyme® further improved the two parameters to 662 mg kg-1 and 36.94%, respectively. The permeate of the hydrolysate after ultrafiltration through a 5 kDa membrane could be effectively decolored by the macroporous resin XAD-16 without notable protein loss, and its LPP content was further boosted to 743 mg kg-1 . CONCLUSION: CGM is a potential new source of LPP and its ultrafiltered and decolored hydrolysate could be used to develop new antihypertensive functional foods. © 2021 Society of Chemical Industry.

L-citrulline enriched fermented milk with Lactobacillus helveticus attenuates dextran sulfate sodium (DSS) induced colitis in mice.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory gastrointestinal diseases that causes worldwide suffering. L. helveticus is a probiotic that can enhance intestinal barrier function via alleviation of excessive inflammatory response. Citrulline, a functional amino acid, has been reported to stimulate muscle synthesis and to function with a prebiotic-like action with certain Lactobacillus strains. The aim of this study was to investigate the potential synergistic effect of combining L. helveticus and citrulline on protection against damage induced by dextran sulfate sodium (DSS) in a mouse model. 6-week-old male C57BL/6J mice were fed with DSS water and randomly divided for administering with different milk treatments: 1) plain milk (control or DSS control), 2) 1% (w/v) citrulline enriched milk (Cit_milk), 3) milk fermented with L. helveticus (LHFM) and 4) DSS+milk fermented with L. helveticus with 1% (w/v) citrulline (Cit_LHFM). The treatment effects on the survival and macroscopic and microscopic signs were examined. All treatments presented different degrees of protective effects on attenuating the damages induced by DSS. All treatments reduced the body weight loss, disease activity index (DAI), histological scores, pro-inflammatory cytokine expression (IL-6, TNF-α and IFN-γ) and production (IL-4) (all P <0.05) and the tight junction (TJ) protein (zonula occluden-1 (ZO-1) expression. LHFM and Cit_LHFM improved survival rate (both at P<0.05). Particularly, Cit_LHFM showed greater effects on protecting the damages induced by DSS, especially in ameliorating colonic permeability, TJ protein (ZO-1, occludin and claudin-1) expression and distribution as well as in reducing IL-4 and IL-17 expression (all P <0.05). Our findings suggested that the combination of and citrulline had significant synergistic effect on protecting against injury from DSS-induced colitis. Therefore, citrulline enriched L. helveticus fermented milk is suggested to be a potential therapy for treating IBD.

BdPUL12 depolymerizes ß-mannan-like glycans into mannooligosaccharides and mannose, which serve as carbon sources for Bacteroides dorei and gut probiotics.

Symbiotic bacteria, including members of the Bacteroides genus, are known to digest dietary fibers in the gastrointestinal tract. The metabolism of complex carbohydrates is restricted to a specified subset of species and is likely orchestrated by polysaccharide utilization loci (PULs) in these microorganisms. ß-Mannans are plant cell wall polysaccharides that are commonly found in human nutrients. Here, we report the structural basis of a PUL cluster, BdPUL12, which controls ß-mannan-like glycan catabolism in Bacteroides dorei. Detailed biochemical characterization and targeted gene disruption studies demonstrated that a key glycoside hydrolase, BdP12GH26, performs the initial attack on galactomannan or glucomannan likely via an endo-acting mode, generating mannooligosaccharides and mannose. Importantly, coculture assays showed that the B. dorei promoted the proliferation of Lactobacillus helveticus and Bifidobacterium adolescentis, likely by sharing mannooligosaccharides and mannose with these gut probiotics. Our findings provide new insights into carbohydrate metabolism in gut-inhabiting bacteria and lay a foundation for novel probiotic development.

Functional characterization and immunomodulatory properties of Lactobacillus helveticus strains isolated from Italian hard cheeses.

Lactobacillus helveticus carries many properties such as the ability to survive gastrointestinal transit, modulate the host immune response, accumulate biopeptides in milk, and adhere to the epithelial cells that could contribute to improving host health. In this study, the applicability as functional cultures of four L. helveticus strains isolated from Italian hard cheeses was investigated. A preliminary strain characterization showed that the ability to produce folate was generally low while antioxidant, proteolytic, peptidase, and ß-galactosidase activities resulted high, although very variable, between strains. When stimulated moDCs were incubated in the presence of live cells, a dose-dependent release of both the pro-inflammatory cytokine IL-12p70 and the anti-inflammatory cytokine IL-10, was shown for all the four strains. In the presence of cell-free culture supernatants (postbiotics), a dose-dependent, decrease of IL-12p70 and an increase of IL-10 was generally observed. The immunomodulatory effect took place also in Caciotta-like cheese made with strains SIM12 and SIS16 as bifunctional (i.e., immunomodulant and acidifying) starter cultures, thus confirming tests in culture media. Given that the growth of bacteria in the cheese was not necessary (they were killed by pasteurization), the results indicated that some constituents of non-viable bacteria had immunomodulatory properties. This study adds additional evidence for the positive role of L. helveticus on human health and suggests cheese as a suitable food for delivering candidate strains and modulating their anti-inflammatory properties.

Proteolytic activity of selected commercial Lactobacillus helveticus strains on soy protein isolates.

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.

Biotransformation of aflatoxin B1 by Lactobacillus helviticus FAM22155 in wheat bran by solid-state fermentation.

Lactobacillus helveticus FAM22155 was the most efficient among five lactic acid bacteria at removing aflatoxin B1 (AFB1) during solid-state fermentation on wheat bran substrate. The mechanism of removal was explored by comparing different fermentation modes. Liquid fermentation had little effect on the breakdown of AFB1. However, a protein extract from the fermented bran was equally effective at degrading aflatoxin B1 as living cell digestion. After treatment with heat and protease K, the degrading capacity of the protein extract was significantly reduced. Taken together, the observed biotransformation of AFB1 was mainly associated with proteins produced during bran fermentation. Four products of U-[13C17]-AFB1 were found by mass spectrometry, including Ⅱ-1 (C11H10O4), Ⅱ-2 (C11H10O4), III (C15H12O5), and IV (C14H10O4). These products all lack the lactone ring indicating lower toxicity than aflatoxin B1.

Acetate kinase and peptidases are associated with the proteolytic activity of Lactobacillus helveticus isolated from fermented food.

Lactobacillus (L.) helveticus is widely used in food industry due to its high proteolytic activity. However, such activity varies greatly between isolates, and the determining factors regulating the strength of proteolytic activity in L. helveticus are unclear. This study sequenced the genomes of 60 fermented food-originated L. helveticus and systemically examined the proteolytic activity-determining factors. Our analyses found that the strength of proteolytic activity in L. helveticus was independent of the isolation source, geographic location, phylogenetic closeness between isolates, and distribution of cell envelope proteinases (CEPs). Genome-wide association study (GWAS) identified two genes, the acetate kinase (ackA) and a hypothetical protein, and 15 single nucleotide polymorphisms (SNPs) that were associated with the strength of the proteolytic activity. Further investigating the functions of these gene components revealed that ackA and two cysteine peptidases coding genes (pepC and srtA) rather than the highly heterogeneous and intraspecific CEPs were linked to the level of proteolytic activity. Moreover, the sequence type (ST) defined by SNP analysis revealed a total of ten STs, and significantly weaker proteolytic activity was observed among isolates of ST2. This study provides practical information for future selection of L. helveticus of strong proteolytic activity.

Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.

Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI®) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

Anticancer potential of an exopolysaccharide from Lactobacillus helveticus MB2-1 on human colon cancer HT-29 cells via apoptosis induction.

This study aimed at investigating the anticancer activity of an exopolysaccharide (EPS) isolated from Lactobacillus helveticus MB2-1. The crude EPS from L. helveticus MB2-1 (LHEPS) was fractionated into three fractions, namely LHEPS-1, LHEPS-2 and LHEPS-3. LHEPS-1 exhibited the most effective anti-proliferative activity, which was associated with a stronger inhibition rate and increased lactate dehydrogenase leakage of human colon cancer HT-29 cells. Flow cytometry analysis and colorimetric assay revealed that LHEPS-1 induced cell cycle arrest by preventing G1 to S transition and increased the apoptosis rate. Furthermore, LHEPS-1 enhanced the production of intracellular reactive oxygen species (ROS) and the activity of caspases-8/9/3, increased the levels of pro-apoptotic Bax and mitochondrial cytochrome c, while decreased the anti-apoptotic Bcl-2 level, indicating that LHEPS-1 might induce the apoptosis of HT-29 cells through a ROS-dependent pathway and a mitochondria-dependent pathway. These findings suggest that LHEPS-1 may be developed as an effective food and/or drug for the prevention and therapeutics of cancer, especially human colon cancer.

Determination of native amino acids and lactic acid inLactobacillus helveticusculture media by capillary electrophoresis using Cu2+and ß-cyclodextrins as additives.

A capillary electrophoresis method for selective simultaneous determination and quantitation of native amino acids and lactic acid during cultivation of Lactobacillus helveticus D75 and D76 strains on the MRS-1 and milk nutrient media was presented. The method provided sensitive UV-detection of native analytes with minimum sample preparation and appeared to be extremely useful for the analysis of culture media. Native amino acids and lactic acid were separated and detected as complexes with Cu2+ ions, while proposed application of ß-cyclodextrin (ß-CD) and its charged and uncharged derivates (sulfated ß-CD and 2-hydroxypropyl-ß-CD) as pseudo stationary phases provided better separation selectivity. The effect of CDs, Cu2+, sodium acetate, ß-CDs concentrations and pH of background electrolyte (BGE) on the electrophoretic mobilities of AAs was thoroughly investigated. The composition of the BGE was found to be as follows: 20 mM acetate buffer solution, 50 mM CuSO4, 10 mM 2-hydroxypropyl-ß-CD, pH 4.3. The developed method possessed high analysis-to-analysis and day-to-day repeatability of migration times (RSD ≤ 1.0% and ≤ 2.5%, respectively). The differences in production of amino acids by D75 and D76 strains grown together and separately were found and concluded to be a consequence and/or one of the causes of synergism and syntropy of the strains. The developed method proved to be applicable for the analysis of culture media.

Lactobacillus helveticus-Fermented Milk Whey Suppresses Melanin Production by Inhibiting Tyrosinase through Decreasing MITF Expression.

Whey obtained from milk fermented by the Lactobacillus helveticus CM4 strain (LHMW) has been shown to improve skin barrier function and increase skin-moisturizing factors. In this study, we investigated the effects of LHMW on melanin production to explore the additional impacts of LHMW on the skin. We treated mouse B16 melanoma cells with α-melanocyte-stimulating hormone (α-MSH) alone or simultaneously with LHMW and measured the amount of melanin. The amount of melanin in B16 cells treated with α-MSH significantly increased by 2-fold compared with that in control cells, and tyrosinase activity was also elevated. Moreover, treatment with LHMW significantly suppressed the increase in melanin content and elevation of tyrosinase activity due to α-MSH. LHMW also suppressed the α-MSH-induced increased expression of tyrosinase, tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) at the protein and mRNA levels. Furthermore, the mRNA and protein microphthalmia-associated transcription factor (MITF) expression levels were significantly increased with treatment with α-MSH alone, which were also suppressed by LHMW addition. LHMW suppression of melanin production is suggested to involve inhibition of the expression of the tyrosinase gene family by lowering the MITF expression level. LHMW may have promise as a material for cosmetics with expected clinical application in humans.

High protein yogurt with addition of Lactobacillus helveticus: Peptide profile and angiotensin-converting enzyme ACE-inhibitory activity.

In order to evaluate differences in the peptide profile and bioactive potential in dairy products, by increasing the protein content and using proteolytic bacteria strain to enable the release of bioactive peptides, a high-protein yogurt with adjunct culture was developed. The effect of protein content, the addition of Lactobacillus helveticus LH-B02, and storage time were evaluated. The qualitative analysis of peptide profile was performed using a mass spectrometry approach (MALDI-ToF-MS), and the potential bioactivity evaluated by ACE inhibition activity. Protein content did not affect the peptide profile in yogurts, and the addition of Lactobacillus helveticus LH-B02 favored the formation of peptides recognized as bioactive, such as αS1-CN f(24-32) and ß-CN f(193-209). Increased protein content and adjunct culture addition increased the ACE inhibitory activity. The combination of both factors had no additional effect on the bioactive potential of yogurts.