Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of β‐galactosidase by Kluyveromyces lactis

The aim of this study was to enhance β‐galactosidase production by Kluyveromyces lactis CICC1773. Firstly, the optimum culture conditions were obtained by response surface methodology, and the maximum β‐galactosidase activity reached 20.6 U mL^?1, about two‐fold increase than that of the initial conditions (initial fermentation medium and conditions). To further improve β‐galactosidase production, a new fed‐batch strategy based on pH feedback control was developed successfully in a 7‐L fermenter, using 400 g L^?1 lactose as feeding medium. As a result, the β‐galactosidase activity and productivity reached up to 111.61 U mL^?1 and 5.31 U/(mL·h), enhanced by 15.3‐fold and 17.6‐fold superior than the results of initial conditions, respectively. To our knowledge, β‐galactosidase activity obtained was the highest value among the results reported by nonrecombinant strains. These results demonstrated that the new fed‐batch strategy based on optimum culture conditions could be automatic control easily and be conductive to further scale up for industrial fermentation.     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.     

Effect of Mentha spicata L. and Mentha aquatica L. essential oils on the microbiological properties of fermented dairy product,kashk

The antibacterial activity of Mentha spicata and Mentha aquatica essential oils (EO) was tested against Staphylococcus aureus, Lactobacillus reuteri, Bifidobacterium animalis and Clostridium perfringens using agar well and disc diffusion techniques. Results showed that M. spicata EO had the highest inhibition activity against the studied microorganisms. Then, the antibacterial activity of both EO at 1500 and 2500 ppm was examined in industrial liquid kashk during the storage at 4 °C for 20 days. Both EO reduced the S. aureus viable count below 5 log CFU g^?1 after 4 days; however, the population of C. perfringens, L. reuteri and B. animalis decreased <1 log CFU g^?1 during the storage time. The least deteriorative effect on the lactic acid bacteria was related to M. aquatica. As revealed by organoleptic studies, kashk samples containing M. aquatica EO at 1500 and 2500 ppm were the most preferred samples.     

Microbial transformation of tectoridin from Pueraria flos by Lactobacillus and bifidobacteria

Tectoridin could be hydrolyzed to tectorigenin by β-glucosidase-producing intestinal bacteria. In this study, nine strains of Lactobacillus and bifidobacteria were screened for high levels of β-glucosidase activity. We investigated their ability to transform tectoridin from Pueraria flos to tectorigenin. Lactobacillus reuteri DSM20016 showed the highest cell-envelope associated β-glucosidase activity, whereas the intracellular β-glucosidase activity from Bifidobacterium adolescentis ATCC15703 was higher than the other screened bacterial strains. L. reuteri DSM20016, Lactobacillus rhamnosus GGB41031 and B. adolescentis ATCC15703 showed high bioconversion rate of tectoridin. L. reuteri DSM20016 showed the highest bioconversion efficiency of tectoridin, 100% tectoridin was hydrolyzed and there was an approximate 185-fold increase in the concentration of tectorigenin after 24 h. The present study suggests that L. reuteri DSM20016, L. rhamnosus GGB41031 and B. adolescentis ATCC15703 have great potential for converting tectoridin from Pueraria flos to more bioactive tectorigenin.     

Screening of a natural biodiversity of lactic and propionic acid bacteria for folate and vitamin B12 production in supplemented whey permeate

Lactic acid bacteria (LAB) and propionic acid bacteria (PAB) are known for the production of several important nutraceuticals. We screened 151 LAB and 100 PAB of different origins (fermented foods and feeds) for extracellular folate and intracellular vitamin B12 production in supplemented whey permeate using a standardized microbiological assay (folate) and HPLC (vitamin B12). Five LAB strains belonging to the species Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus brevis and Lactobacillus fermentum exhibited high extracellular folate productions, with a maximum yield of 397 ± 60 ng mL^?1 for L. plantarum SM39. The highest vitamin B12 production was measured for Propionibacterium freudenreichii DF15 with 2.5 μg mL^?1. Screening a large biodiversity of LAB and PAB led to a representative image of the distribution of folate and vitamin B12 production by these genera and enabled the identification of high natural folate and vitamin B12 producing strains with high potential for applications in fermented foods.     

Reduction of Listeria monocytogenes in cold‐smoked salmon by bacteriophage P100, nisin and lauric arginate,singly or in combinations

Three GRAS antimicrobials including, lauric arginate (LAE), bacteriophage P100 (phage P100) and bacteriocin nisin, were evaluated either singly or in combinations for the reduction of initial load of Listeria monocytogenes in cold‐smoked salmon (CSS). The stability of phage P100 in the presence of LAE (200 ppm) and nisin (500 ppm) or at 10× and 100× of these concentrations was determined at 4 °C or 30 °C for 24 h in a broth model. Phage P100 was found to be highly stable in the presence of these antimicrobial agents as plaque‐forming units (PFU) did not vary between control and antimicrobial‐treated phage. The survival of L. monocytogenes in the presence of phage P100, nisin and LAE showed remarkable reduction within 24 h both at 4 °C or 30 °C in broth. Treatment of CSS containing 3.5 log CFU cm^?2 L. monocytogenes with phage P100 (10⁸ PFU mL^?1), nisin (500 ppm) and LAE (200 ppm) showed strong listericidal action and reduced the L. monocytogenes by 2–3 log CFU cm^?2 after 24 h. Among the combined treatments, phage P100 + LAE or nisin + LAE exhibited the most listericidal action in which L. monocytogenes cells were reduced to undetectable level within 24 h in CSS.     

Chaperone‐like activity of β‐casein and its effect on residual in vitro activity of horseradish peroxidase

In this study, the residual activity horseradish peroxidase was used as a novel marker of chaperone‐like activity of β‐casein under elevated temperature. It was shown that β‐casein does affect residual activity of horseradish peroxidase (HRP) depending on the concentration and molar ratio between proteins. Incubating HRP (0.1 mg mL^?1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL^?1 β‐casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β‐casein on HRP disappears when pH is below the isoelectric point of β‐casein. It was also proven by light scattering studies that β‐casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries such as enzyme manufacturers and food industry.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Mixed fermentation of jujube juice (Ziziphus jujuba Mill.) with L. rhamnosus GG and L. plantarum-1: effects on the quality and stability

This study was conducted aiming at improving the quality of jujube juice by mixed fermentation of lactobacilli. Selection of favourable lactobacilli and addition of nitrogen sources were explored as an efficient method to improve the viability of probiotic in jujube juice. After fermentation, the viability increased to 9.15 ± 0.10 Log CFU mL⁻¹, while the content of lactic acid increased to 5.61 ± 0.03 mg mL⁻¹. The total phenolic and total flavonoid content were 2663.03 ± 11.95 μg mL⁻¹ and 163.95 ± 0.47 μg mL⁻¹ respectively. Moreover, the stability of fermented jujube juice during refrigeration was investigated, which showed that the viability dropped to 8.84 ± 0.6 Log CFU mL⁻¹ and the concentration of lactic acid slowly increased to 6.51 ± 0.04 mg mL⁻¹; the ABTS value showed a 4.26% reduction and FRAP value did not significantly (P < 0.05) change during refrigerated storage. In addition to the existing knowledge, our data aid to the future applications of the jujube as a potential ingredient in novel probiotic foods formulation.     

Antimicrobial activities of high molecular weight water‐soluble chitosans against selected gram‐negative and gram‐positive foodborne pathogens

Antimicrobial activities of high molecular weight water‐soluble chitosans (HMWWS) against selected Gram‐negative and Gram‐positive foodborne pathogens (initial inoculation of ca. 6.5 Log CFU mL^?1) were evaluated. Chitosans with 789 kDa and/or 1017 kDa were dissolved in aspartic acid (AS) to obtain 1–4% w/v solutions. Among HMWWS, only 4% 789 kDa AS chitosan reduced E. coli counts by 2 Log CFU mL^?1 from 7.33 at 0 h to 5.16 Log CFU mL^?1 at 96 h, and they were not effective against S. Typhimurium. Depending on the concentrations, HMWWS completely inhibited V. cholerae, V. vulnificus and V. parahaemolyticus as well as B. cereus and L. monocytogenes after 48 h or 96 h of incubation. Compared with the control (no HMWWS), 2% or 3% 1017 kDa AS chitosans showed about 3 Log CFU mL^?1 lower (4.72–4.86 vs. 7.71) for S. aureus at 96 h of incubation.     

Preparation and optimization of soy protein isolate–high methoxy pectin microcapsules loaded with Lactobacillus delbrueckii

This study was aimed to use soy protein isolate (SPI) and high methoxy pectin (HMP) as encapsulating materials for probiotic bacterial (Lactobacillus delbrueckii) delivery systems. The encapsulation conditions were optimised, and scanning electron microscopy (SEM) was used to characterise the microstructural changes of the microcapsule. The results showed that the optimal conditions for microcapsule preparation were 90 mg mL^?1 SPI and 1 mg mL^?1 HMP, with a SPI/HMP ratio of 7:1 (v/v), and a L. delbrueckii suspension to SPI–HMP complex ratio of 1:1 (v/v). The viability of the probiotics in the microcapsules reaching the small intestine was 3 log CFU mL^?1 higher than that of naked bacteria. SEM showed that the surface of the SPI–HMP compound microcapsules was smooth and that a large number of L. delbrueckii could be seen in cross‐sections of the microcapsules.     

Hazelnut milk fermentation using probiotic Lactobacillus rhamnosus GG and inulin

Following the consumer demand of healthy vegetable products due to their interesting nutritional profiles and potential functionalities, the fermentation process of hazelnut milk with Lactobacillus rhamnosus GG and S. thermophilus was studied. The effect of different factors (glucose, inulin and inoculum contents) was analysed to ensure sufficient probiotic survivals in a minimum time. The shelf life of the optimised product was characterised in terms of its main physicochemical and quality parameters (probiotic survivals and sensory analysis). Results showed that the defined formulation allowed high probiotic survivals (≈10⁸ cfu mL^?1) throughout cold storage and >60% survived to the in vitro digestion process (≈10⁵ cfu mL^?1). Lactobacillus rhamnosus GG was no able to degrade inulin, which remained to exert health benefits in the host. The product was highly appreciated by the sensory panel during its shelf life despite the formation of a weak gel, which presented syneresis at the last storage time.     

Limited hydrolysis of β‐casein by cell wall proteinase and its effect on hydrolysates's conformational and structural properties

Optimisation of enzymatic hydrolysis of β‐casein with cell envelope proteinase (CEP) from Lactobacillus acidophilus JQ‐1 to produce the angiotensin‐I‐converting enzyme (ACE) inhibitory peptides using response surface methodology (RSM). Under optimal conditions (enzyme‐to‐substrate ([E]/[S]) ratio (w/w) of 0.132 and pH of 8.00 at 38.8 °C), the ACE inhibitory activity of hydrolysates was 72.06% and the total peptides was 11.75 mg mL^?1. Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β‐casein surface structure was gradually weakened and small holes appeared after enzymatic treatment, while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in the chemical composition and macromolecular conformation of β‐casein after enzymatic hydrolysis. Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates had higher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfhydryl content of β‐casein hydrolysates compared with raw β‐casein, which led to the increase in the antioxidant activity of β‐casein hydrolysates.     

Membrane‐disruptive property of a novel antimicrobial peptide from anchovy (Engraulis japonicus) hydrolysate

A novel antimicrobial peptide named Pep39 has been isolated from anchovy hydrolysate using ÄKTA protein liquid chromatography and reverse‐phase high‐performance liquid chromatography. Its amino acid sequence was RLFRHAFKAVLRL with a molecular weight of 1626.92. Pep39 demonstrated antimicrobial ability against Escherichia coli ATCC 25922, Salmonella typhi ATCC 50013, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 9372, with minimal inhibitory concentration (MIC) ranging from 4 to 32 μg mL^?1. No cytotoxicity to mouse red blood cells was observed when its concentration was lower than 30 μg mL^?1. Pep39 induced the influx of fluorescent probe 8‐Anilino‐1‐naphthalenesulfonic acid and the outflow of β‐galactosidase by increasing E. coli outer and inner membrane permeabilities, respectively. Flow cytometric analysis also demonstrated that Pep39 disrupted E. coli cell membrane. These results suggested that the antimicrobial mechanism of Pep39 involved cytoplasmic membrane damage.     

Enzymatic grafting of gallate ester onto chitosan: evaluation of antioxidant and antibacterial activities

The antioxidant octyl gallate (OG) has been successfully grafted onto chitosan by means of horseradish peroxidase biocatalyst. The maximum gallate incorporation onto chitosan determined by ¹H NMR spectroscopy was up to 16 molar%. The resulting materials displayed antioxidant capacities with DPPH radical inhibition percentage up to 23% upon the assay conditions. The grafting of the antioxidant on chitosan enhanced its antimicrobial activity. Minimal inhibitory concentration (MIC) for Escherichia coli was 0.5 g L^?1. The native medium molecular weight chitosan alone or grafted with the lowest OG incorporation (ca. 11 molar%) attained 100% inhibition of E. coli, whereas lower inhibition was observed for all other materials 50.7–68.9% corresponding a reduction in the counts from 10.6 to 5.23–3.30 Log CFU mL^?1. The inhibition of Listeria monocytogenes was significantly higher (59.8–100%) than that with the Gram negative bacterium reaching the MIC at 0.25 g L^?1 with a reduction in the counts from 12.6 Log CFU mL^?1 to 5.06–0 Log CFU mL^?1.     

Effect of pomegranate (Punica granatum) pomace extract as a washing agent on the inactivation of Listeria monocytogenes inoculated on fresh produce

Pomegranate pomace extract (PPE) was used as a substitute to chlorinated water for washing of fresh produce. PPE contained punicalagins and ellagic acids, and it had a good inhibitory effect against Listeria monocytogenes based on the results of disc diffusion test and time‐kill assay. Red chard, beet, chicon and red mustard leaves were treated with 7 mg mL^?1 PPE as a washing solution to inactivate L. monocytogenes inoculated on fresh produce and compared with 0.05 mg mL^?1 NaOCl treatment. After the treatment, L. monocytogenes population on the four leafy vegetables was reduced by 1.96–2.97 log CFU g^?1 compared to that on nontreated samples. The reductions in L. monocytogenes after PPE treatment were higher than those observed after NaOCl treatment. Further, the PPE treatment did not affect the surface colour of all fresh produce applied in this study. Thus, PPE can be used as an effective antimicrobial washing agent of fresh produce.     

Innovative production of multistrain synbiotic product using Thai-pigmented rice and rice bran oil

This study found that the isolated probiotics Lactobacillus paracasei KUKPS6201, L. acidophilus KUKPS6107, L. reuteri KUKPS6103, L. rhamnosus KUKPS6007, L. salivarius KUKPS6202, Bacillus coagulans KPSTF02 and Saccharomyces boulardii KUKPS6005 had high potential for probiotic properties. All strains had antibacterial activity and high antioxidant activity of 1.654 ± 0.017 mg Trolox mL⁻¹ probiotic extract. The selected strains could survive in a simulated gastrointestinal tract under anaerobic conditions and showed no haemolytic activity. Furthermore, the probiotic strains were strongly auto-aggregated and also showed co-aggregated ability with pathogenic bacteria. The probiotic microorganisms demonstrated high ability to adhere to Thai-pigmented rice grains. The results of analysis of these probiotics showed that Riceberry rice bran oil was an excellent prebiotic. A synbiotic product containing Thai-pigmented rice grains (cultivar Riceberry, Luem Pua and Black Jasmine) and rice bran oil was produced. After 8 weeks of storage, the viability of the probiotics in terms of multistrains was 7.36 ± 0.04 log CFU g^–1 (85.78% survival rate). Microbiological safety testing indicated that the amounts of contaminants were acceptable. This study provided the first scientific report on the feasibility of applying Thai-pigment rice, rice bran oil and mixed-culture probiotics as a novel functional synbiotic product.     

Use of banana purée from three indigenous Thai cultivars as food matrices for probiotics and application in bio‐set‐type yoghurt production

This research studied the use of banana purée from three indigenous Thai cultivars (Kluai Hom Thong, Kluai Namwa and Kluai Khai) as probiotic carrying agents in food and application in bio‐set‐type yoghurt. Kluai Khai purée had promising indication of prebiotic activity on Lactobacillus paracasei Lpc‐37 with the highest probiotic activity score. When Lpc‐37 cell was incorporated into Kluai Khai purée, its cell counts increased by 0.8 log CFU g^?1 and it adhered to purée, as seen by the proliferating cells. Therefore, Kluai Khai purée mixed with Lpc‐37 was selected for the production of bio‐set‐type yoghurt. The results showed that viable cells increased more than 0.8 log CFU g^?1 during storage. This study reflected the achievement of using Kluai Khai purée as food matrix for carrying ‘active probiotic cells’ in bio‐yoghurt production. This methodologically developed prototype could be an interesting alternative for value adding of indigenous fruits.     

Control of lactic acid bacteria in fermented beverages using lysozyme and nisin: test of traditional beverage boza as a model food system

The objective of this study was to increase quality and limited shelf‐life of boza (3–15 days), a traditional Balkan origin fermented beverage using lysozyme (LYS) and/or nisin (NIS). For this purpose, the effectiveness of NIS, LYS and LYS:NIS combinations was first tested in a broth medium at 4 °C for 3 weeks on Lactobacillus plantarum, one of the frequently isolated lactic acid bacteria in boza. Stability of LYS and NIS in boza, their effects on LAB counts, and chemical and sensory properties of boza were then evaluated during cold storage at 4 °C. Results of LAB counts as well as pH, d ‐ and l ‐lactic acid, and titratable acidity measurements showed that LAB in boza containing NIS (250 μg g^?1) or LYS:NIS (500:250 μg g^?1) could be controlled without reducing LAB counts below 6 log CFU mL^?1 during 2 weeks shelf‐life. In contrast, LYS (500 μg g^?1) alone could not control LAB in boza to delay its acidic spoilage. Positive effects of NIS and LYS:NIS application on quality of boza were also proved with sensory analysis by panelists and e‐nose measurements. This work showed that use of natural GRAS agents in preservation of fermented beverages containing probiotic LAB is possible without affecting their characteristic aroma and flavour.     

Preparation and characteristics of squid pen β‐chitin prepared under optimal deproteinisation and demineralisation condition

Squid (Todarodes pacifica) pen was an excellent source of β‐chitin with 25.5% yield. The optimal condition to prepare squid pen β‐chitin was established: deproteinisation with 3% NaOH for 30 min at 15 psi/121 °C and a solid/solvent ratio of 1:10 (w/v) and a subsequent demineralisation with 1 N HCl for 30 min at room temperature and a solid/solvent ratio of 1:10 (w/v). Squid pen β‐chitin contained 6.29% nitrogen, 0.25% ash, and negligible fat with degree of acetylation of 94.02%, residual amino acid of 0.499 g/100 g and bulk density of 0.28 g mL^?1. Depending on its particle size, squid pen β‐chitin visually looked white (L* = 82.82, a* = ?0.67, b* = 6.31; particle size of 0.15–0.18 mm) or light grey (L* = 62.88, a* = 0.33, b* = 10.66; particle size of 0.425–0.841 mm). Water, fat and dye‐binding capacity of squid pen β‐chitin was 694.67%, 194.03% and 79.81%, respectively.