Slovak Cheese Research Articles

Analysis of Mycotoxin and Secondary Metabolites in Commercial and Traditional Slovak Cheese Samples.

Cheese represents a dairy product extremely inclined to fungal growth and mycotoxin production. The growth of fungi belonging to Aspergillus, Penicillium, Fusarium, Claviceps, Alternaria, and Trichoderma genera in or on cheese leads to undesirable changes able to affect the quality of the final products. In the present investigation, a total of 68 types of commercial and traditional Slovak cheeses were analyzed to investigate the occurrence of fungal metabolites. Altogether, 13 fungal metabolites were identified and quantified. Aflatoxin M1, the only mycotoxin regulated in milk and dairy products, was not detected in any case. However, the presence of metabolites that have never been reported in cheeses, such as tryptophol at a maximum concentration level from 13.4 to 7930 µg/kg (average: 490 µg/kg), was recorded. Out of all detected metabolites, enniatin B represents the most frequently detected mycotoxin (0.06–0.71 µg/kg) in the analyzed samples. Attention is drawn to the lack of data on mycotoxins’ origin from Slovak cheeses; in fact, this is the first reported investigation. Our results indicate the presence of fungal mycotoxin contamination for which maximum permissible levels are not established, highlighting the importance of monitoring the source and producers of contamination in order to protect consumers’ health.

Aroma profile and lactic acid bacteria characteristic of traditional Slovak cheese "May bryndza".

This paper describes the results of the characterization of a traditional Slovak cheese called "May bryndza" with regard to the profiles of volatile organic compounds and lactic acid bacteria. Samples of "May bryndza" cheese produced solely from unpasteurized ewe's milk were collected from 4 different Slovak farms, and samples of the cheese produced from a mixture of 2 types of milk (raw ewe's and pasteurized cow's milk) were collected from 3 different Slovak industrial dairies. There were 15 compounds detected and identified by the electronic nose. The impact of the kind of milk and the kind of dairy on the aroma profile of the product was not confirmed by PCA. The compounds with the highest relative contents in samples were acetoin (2.59%-24.55%), acetic acid (6.69%-13.39%), methoxy-phenyl-oxime (4.49%-8.52%), butanoic acid (1.89%-5.67%), and 2,3-butanediol (0.98%-4.08%), which were determined with gas chromatography. A total of 1533 isolates of LAB were obtained from the "May bryndza" cheese samples. Four families, five genera, and 19 species were identified with mass spectrometry, and isolated bacteria, both from the farm and industry dairies were the most frequently found to belong to Lactococcus lactis subsp. lactis.

Diversity of microbiota in Slovak summer ewes' cheese "Bryndza".

“Bryndza” cheese is an important Slovak traditional regional product. New knowledge on the role of microorganisms involved the “Bryndza” ripening process may provide valuable data on its quality and safety. In our study, the “Bryndza” made from pasteurized ewes milk was studied towards total count of bacteria, coliforms bacteria, enterococci, lactic acid bacteria, and microscopic filamentous fungi. All those groups of microbiota were detected using classical microbiological methods and identified using mass spectrometry. A total of 3,758 isolates were identified with score higher than 2.00. Altogether, 13 families, 24 genus, and 44 species of microbiota were identified in Slovak cheese “Bryndza.” The most often isolated species were yeasts Yarrowia lipolitica and Dipodascus geotrichum and the lactic acid bacteria Lactobacillus paracasei subsp. paracasei.

Detection of adulteration of traditional Slovak bryndza ewe’s cheese with cow’s lump cheese by isoelectric focusing of gamma caseins

ABSTRACT “Slovak bryndza” is a traditional Slovak cheese manufactured by milling a lump of matured ewe’s cheese or by milling a mixture of lump ewe’s cheese and lump cow’s cheese. The percentage of lump ewe’s cheese is greater than 50% w/w dry matter. Differences in the purchase prices of ewe’s and cow’s milk create the potential for adulteration. The detection of cow’s milk compounds by isoelectric focusing of γ-caseins after plasmolysis is a suitable method for the determination of adulteration of this traditional cheese. The analysis and final results may be affected by factors affecting laboratory activity and biological factors. Biological factors are manifested primarily as variations in the casein content of ewe’s and cow’s milk. Despite this variation, this method may also be used for quantification purposes. We were able to establish a reliable calibration line from the internal reference materials prepared in our laboratory. The mean composition of Slovak bryndza cheese determined in our research was protein 18.7 ± 1.9 g 100 g−1, fat 24.0 ± 1.8 g 100 g−1, lactose 1.5 ± 1.9 g 100 g−1, ash 4.5 ± 0.3 g 100 g−1, fat in dry matter 49.3 ± 2.6 g 100 g−1, casein 18.4 ± 1.9 g 100 g−1, gamma casein 0.60 ± 0.19 g 100 g−1, and bovine-to-ovine gamma casein ratio 0.65 ± 0.12 g 100 g−1.

Preference mapping of Slovak cheese

The production of steamed cheese has a long tradition in Slovakia. Some of these cheeses have even received the PGI designation, which is a designation granted to products with specific geographical characteristics (Slovenská parenica, Zázrivské vojky, Zázrivský korbáčik, and Oravský korbáčik). In our study, eight samples of various unsmoked steamed cheese from small dairy farms (samples D, F, G, and H) and medium-sized farms (A, B, C, and E) were evaluated. Our work aimed to determine whether there are significant differences in sensory characteristics between samples from small and medium-sized dairy farms and whether there are differences in the preferences between these samples for consumers. Samples were evaluated by sensory analysis, where the assessors evaluated the characteristics of color, odor, texture, flavor, and overall appearance on a nine-point hedonic scale. Differences at a statistically significant level in the attributes of odor, texture, flavor, and overall appearance were confirmed (p <0.05) between samples from small and medium dairy farms, no statistically significant difference was proved in the attribute color (p >0.05). The results were processed using PCA, whereas can be seen from the graphic representation the carriers of all evaluated attributes were samples from medium-sized farms (except for sample D, which belonged to the first group of samples together with samples from medium-sized farms). We constructed a preferential map by combining internal and external mapping, while the internal data were formed by data obtained from assessors using sensory analysis and the external data came from an online questionnaire. Using the preferential mapping technique, we found out that samples from medium-sized dairy farms were classified as the most preferred samples which correspond with the results of sensory analysis.

The characteristic of sheep cheese “Bryndza” from different regions of Slovakia based on microbiological quality

The aim of our study was to describe microorganisms which occur in the traditional Slovak cheese „Bryndza“. There were a total of 60 cheese samples collected from ten different farms during May 2019. The microbiota studies included the total bacterial count, coliforms, enterococci, lactic acid bacteria, yeasts and microscopic fungi. The total bacterial counts were cultivated on plate count agar at 30 °C in aerobic conditions, lactic acid bacteria on MRS at 37 °C in anaerobic conditions, coliform on VRBL and VRBG at 37 °C in aerobic condition, yeasts and microscopic fungi on MEA at 25 °C under aerobic condition. Gram-positive, Gram-negative and yeasts isolates were identified with MALDI-TOF MS Biotyper. Totally, a number of 1175 isolates of G-, G+ and yeast were identified with score higher than 2 and moulds. Escherichia coli and Stenotrophomonas maltophilia were the most frequently identified species of Gram-negative and Leuconostoc mesenteroides ssp. mesenteroides and Lactococcus lactis ssp. lactis from Gram-positive bacteria. Yarrowia lipolitica and Kluyveromyces lactis were the most distributed yeasts. Lactic acid bacteria group was represented by Lactobacillus, Lactococcus, Leuconostoc and Pediococcus. The most abundant genera of lactic acid bacteria were Lactobacillus with 11 species. This study describes the indigenous microbiota of the traditional ewe's milk cheeses from Slovakia.

Microbiota of Non-Smoked Slovak Cheese “Parenica”

Abstract Our study was focused on rapid identification of selected groups of microorganisms from non-smoked cheese made from cow’s milk. The following groups of microorganisms were detected: lactic acid bacteria, total microbial counts, coliforms, yeast and filamentous microscopic fungi. The microbial groups were analyzed depending on sampling month from January to December. The microbial qualtity of samples were evaluated with classical microbiological method and than identified with mass spectrometry. The highest total microbial count was found in July - 3.42 log CFU/g. The presence of coliform bacteria was detected in three samples in the beginning of January and the counts increase in summer months. Microscopic filamentous fungi and yeast were present in all samples during the year with the highest counts during the summer months were observed. Altogether, 25 microbial genera were identified including families Candida, Cryptococcus, Kluyveromyces, Saccharomyces, Rhodotorula, Torulaspora, Yarrowia, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Escherichia, Klebsiella, Hafnia, Citrobacter, Bacillus, and Sphingomonas with mass spectrometry method. The lactic acid bacteria are one of the most important group of microorganisms in milk and milk products for production of typical sensory characteristics.

Effect of somatic cell counts occurred in milk on quality of Slovak traditional cheese - Parenica

The aim of this work was to compare somatic cell count in milk used for making steamed cheese Parenica in Slovak industrial dairies and small farm dairies and to find out whether somatic cell counts in milk affect the dry matter content of Parenica cheese. The samples of raw milk were taken from 3 industrial dairies (A, B, C) and from 3 farm dairies (E, F, G), produced traditional Slovak cheese Parenica in period from January untill December 2018. The somatic cell count in milk was determined by FossomaticTM 5000 (Foss, Denmark) and dry matter of cheese by oven drying method to constant weight. There were no statistically significant differences (p >0.05) for somatic cell counts in milk processed in industrial and farm dairies. Lower somatic cell counts were determined in milk amples from industrial dairies (mean value 326.55 thousand in 1 mL) in comparison to milk samples from farm dairies (mean value 507.67 thousand in 1 mL). Statistically lower dry matter content (p <0.01) in the samples of Parenica cheese was found out in farm dairy E in comparison to other dairies. The relationship between somatic cell count in milk and dry matter in cheese was confirmed by the relatively low correlation coefficients in dairies, A = 0.22; C = 0.15 and F = -0.12 and higher correlation coefficients in dairies, B = -0.32; D = 0.45 and E = -0.48. Obtaining a more accurate effect of somatic cell count on cheese quality requires the continuation of the research on a larger number of samples and consideration of other factors.

Diversity of microorganisms in the traditional Slovak cheese

The aim of the present study was to describe the microbial groups of the traditional Slovak cheese Parenica during rippening. The microbial group included the total bacterial count, coliform bacteria, enterococci, lactic acid bacteria, and microscopic filamentous fungi, which may affect the organoleptic characteristics of this product. A total of 42 cheese samples were collected from four different farms during three months. The total bacterial counts were cultivated on Plate count agar at 30 °C, lactic acid bacteria (LAB) on MRS, APT and MSE at 37 °C, coliform bacteria on VRBL at 37 °C. Gram-positive and Gram-negative isolates were identified by MALDI-TOF MS profiling. Bacillus sp. and Enterococcus faecium were the most frequently identified species of bacteria. Candida kefyr was the most distributed yeast according to microbiological methods. Lactic acid bacteria group was represented by Lactobacillus helveticus, L. jensenii, L. alimentarius, L. crispatus, L. curvatus, L. fermentum, L. suebicus, L. delbrueckii ssp. lactis, L. paracasei ssp. paracasei, Lactococcus lactis ssp. lactis, Leuconostoc lactis and Le. mesenteroides ssp. mesenteroides . This report describing the indigenous microbiota of the traditional raw milk cheeses from Slovakia. Our results provide useful information on occurrence of valuable microbial strain for the industrialization of producing of the traditional dairy products in Slovakia.

APPLICATION OF MALDI-TOF MASS SPECTROMETRY FOR IDENTIFICATION OF BACTERIA ISOLATED FROM TRADITIONAL SLOVAK CHEESE “PARENICA”

In this study, the cultivable population of bacteria from a traditional Slovak cheese “Parenica” made from cow milk were identified using MALDI-TOF mass spectrometry (MS). A total of 100 “Parenica” cheese packages from four producers were examined, including n=50 smoked and n=50 non-smoked cheese samples. The lactic acid bacteria (LAB) were cultured on MRS, APT and MSE agars at 30 °C, coliform bacteria on VRBL agar at 37 °C, total count of bacteria (TCB) on Plate count agar at 30 °C and enterococci on Enterococcus selective agar at 37 °C. Gram-positive and gram-negative strains were subjected to identification by MALDI-TOF MS profiling. MALDI-TOF MS identification revealed four genera belonging to LAB including Lactococcus, Lactobacillus, Enterococcus and Leuconostoc. Lactobacillus was the most represented genus with seven species: Lactobacillus curvatus (L. curvatus), L. delbrueckii, L. fermentum, L. casei, L. paraplantarum, L. plantarum and L. sakei.. Escherichia coli, Enterobacter asburiae, Klebsiella oxytoca and K. pneumoniae were the most identified bacteria species from Enterobacteriales order.

Application of electronic nose for determination of Slovak cheese authentication based on aroma profile

Electronic nose with sensors is used in many industries and for various applications such as quality control, process monitoring, shelf life evaluation, origin or authenticity assessment. The aim of this work was to investigate the electronic nose with FID detectors applicability for characterization of steamed cheese and for the assessment of steamed cheese quality decay during storage. Samples of smoked and unsmoked steamed cheese varieties from 5 Slovak enterprises concerning different regions of Slovakia were analysed. Data from aroma profiles were processed by statistical technique PCA. Compounds like acetaldehyde, 1-propanal, propanoic acid, ethyl hexanoate, furfural, butan-2-one, isovaleric acid, 1-hexanol or α-pinene were determined as significant flavours in fresh steamed cheese samples. In the current study, no significant differences in aroma profiles between fresh and stored cheese samples were confirmed. Thus, differences in main odour substances composition of steamed cheese varieties, obtained from various producers in several geographic regions of Slovakia, were minor.

The genome sequence of Enterococcus faecium 8S3, lactic acid–producing bacterium from Slovak cheese – bryndza with biotechnological potential

New genetic data with biotechnological potential (citrate metabolism, proteases, bacteriocin production) provides the genome sequence of the lactic acid producing bacterium of Enterococcus faecium strain 8S3, isolated from traditional Slovak cheese - bryndza produced from unpasteurised ewe milk. The genome sequence consists of 2.8 Mbp, with a mean G + C content of 38.2% and show high similarity to other E. faecium genome sequences. A total of 2.833 coding sequences, including 62 structural RNAs (3 rRNA and 59 tRNA) were predicted. Comparative genomic data indicate that prophages and bacteriophage remnants are the main source of diversity among E. faecium genomes.

Identification of the Slovak traditional cheese “Parenica” microflora

Numerous studies have demonstrated the higher accuracy, faster time-to-results and lower costs provided by MALDI Biotyper systems compared to classical methods. In this study, the culturable population of total count of bacteria, enterococci, coliforms bacteria, lactic acid bacteria (LAB) and microscopic fungi and yeasts from cow’s dairy products was identified using the MALDI-TOF MS Biotyper. Altogether, 50 samples of the Slovak cheese “Parenica” were examined. Total numbers of bacteria were cultured on Plate count agar at 37 °C for 24–48 h, aerobically; enterococci were cultured on Enterococcus selective agar at 37 °C for 24–48 h, aerobically; coliforms bacteria were cultured on Violet Red Bile lactose agar at 37 °C for 24–48 h, aerobically. The LAB were cultured on MRS (Main Rogosa agar), MSE and APT agar at 30 °C in microaerophilic conditions. The microscopic fungi and yeasts were cultured on Malt extract agar at 25 °C for 5 days, aerobically. Isolated strains (total 669) were subjected to identification by the MALDI-TOF MS. Among total count the identified bacteria mostly were Acinetobacter baumannii, Bacillus cereus, Micrococcus luteus and Staphylococcus warneri. Escherichia coli and Enterobacter cloacae were the most abundant coliform bacteria representatives identified. Coliform bacteria included Citrobacter, Hafnia and Klebsiella. Altogether three genera belonged to the LAB – Lactobacillus, Lactococcus and Leuconostoc were identified with Lactococcus lactis, Lactobacillus plantarum, Lactobacillus coryniformis, L. fructivorans and Leuconostoc mesenteroides were considered as the dominated LAB species in dairy products. Among yeasts, Kluyveromyces lactis, Candida zeylanoides and Yarrowia lipolytica were among the most isolated.

Antibiotic Resistance and Prevalence of Enterococcus Spp. and Escherichia Coli Isolated from Bryndza Cheese

The study aimed at determining the prevalence antibiotic resistance of species – identified enterococci and Escherichia (E.) coli isolated from typical fresh Slovak cheese, bryndza. Antibiotic resistance of enterococci was determined by disk diffusion method. Of isolated enterococci, 240 were obtained from bryndza cheese. The first two decimal dilutions from 24 bryndza cheese samples purchased at supermarkets in Košice (0.1 mL) were spread on the surface of Slanetz and Bartley agar and incubated for 48±2 h at 37±1°C. Species identification of enterococci and E. coli was detected by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based on bacterial protein profiling. The following species of enterococci were identified by MALDI-TOF MS: Enterococcus (Ent.) faecalis (22 strains), Ent. faecium (18 strains), Ent. sacharolyticus (6 strains), Ent. gilvus (4 strains), Ent. durans (9 strains), and Ent. casseliflavus (6 strains). All of the 45 E. coli strains and 74 strains of enterococci identified by MALDI-TOF MS were determined for occurrence of blaTEM, blaSHV and blaCTX-M genes. The results of our study suggest that the highest resistance of enterococci was on tetracycline (29.73%) and any resistance was recorded on vancomycin (0%). The highest multidrug-resistance was recorded on two antibiotics (32.43%). Neither one isolate of enterococci was resistant to all 6 antibiotics used in the experiment. In total, 19 (42.22%) E. coli were found to be producers of extended-spectrum β-lactamase.

SURVIVING OF COMMERCIAL PROBIOTIC STRAIN Lactobacillus rhamnosus GG IN SLOVAK COW LUMP CHEESE EXPERIMENTALLY INOCULATED WITH Listeria innocua

Cow lump cheese represents a traditional Slovak cheese. It belongs to fresh types of cheeses. The aim of this study was to test surviving of commercial probiotic strain Lactobacillus rhamnosus GG in cow lump cheese experimentally infected with L. innocua; (listeriae are contaminants) and to check the suitability of GG strain as additive for this product. The counts of GG strain in cow lump cheeses were well balanced during whole experiment. It was found in the counts from 5.48 ± 0.15 to 7.77 ±1.50 log10 cfu/g. Its maximum in cheese was 7.77 ± 1.30 log10 cfu/g on day 7 with stability up to day 14. The identity of GG strain isolated from cheeses was confirmed by PCR. The counts of other lactic acid bacteria were also well balanced during the whole experiment in the experimental cheeses with stability up to day 14. Only in E1/GG cheese, the highest number of LAB was detected (10.60 ±1.26 log10 cfu/g). The count of L. innocua LMG 13568 was not influenced. The pH and lactic acid values were not negatively influenced. Visually, the GG cheese provided a good structure (consistency).It can be disputed that shelf-life of the product could be maintained by this way and/or the product itself with GG strain can be consumed as afunctional food or to serve as a probiotic strain carrier.

Genome Sequence of Lactobacillus plantarum 19L3, a Strain Proposed as a Starter Culture for Slovenska Bryndza Ovine Cheese.

The genome sequence of Lactobacillus plantarum isolated from ovine cheese is presented here. This bacterium is proposed as a starter strain, named 19L3, for Slovenská bryndza cheese, a traditional Slovak cheese fulfilling European Food Safety Authority (EFSA) requirements.

Microbial diversity and dynamics during the production of May bryndza cheese

Diversity and dynamics of microbial cultures were studied during the production of May bryndza cheese, a traditional Slovak cheese produced from unpasteurized ewes' milk. Quantitative culture-based data were obtained for lactobacilli, lactococci, total mesophilic aerobic counts, coliforms, E. coli, staphylococci, coagulase-positive staphylococci, yeasts, fungi and Geotrichum spp. in ewes' milk, curd produced from it and ripened for 0 – 10days, and in bryndza cheese produced from the curd, in three consecutive batches. Diversity of prokaryotes and eukaryotes in selected stages of the production was studied by non-culture approach based on amplification of 16S rDNA and internal transcribed spacer region, coupled to denaturing gradient gel electrophoresis and sequencing. The culture-based data demonstrated an overall trend of growth of the microbial population contributing to lactic acid production and to ripening of the cheese, lactobacilli, lactococci and Geotrichum spp. growing up to densities of 108CFU/g, 109CFU/g and 105CFU/g, respectively, in all three consecutive batches of bryndza cheese. The diversity of bacteria encompassed Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter sp., Acinetobacter johnsonii, Citrobacter braakii, Clostridium bartlettii, Corynebacterium callunae, Corynebacterium maris, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter hormaechei, Enterococcus faecium, Enterococcus pallens, Escherichia coli, Haemophilus haemolyticus, Hafnia alvei, Kluyvera cryocrescens, Lactobacillus helveticus, Lactococcus garvieae, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, “Leuconostoc garlicum”, Mannheimia glucosida, Mannheimia haemolytica, Pseudomonas sp., Ps. fluorescens, “Ps. reactans”, Raoultella ornithinolytica, R. terrigena, “Rothia arfidiae”, Staphylococcus aureus, Staph. epidermidis, Staph. felis, Staph. pasteuri, Staph. sciuri, Staph. xylosus, Streptococcus parauberis, Str. thermophilus and Variovorax paradoxus. The diversity of yeasts and fungi encompassed Alternaria alternata, “Ascomycete sp.”, Aspergillus fumigatus, Beauveria brongniartii, Candida xylopsoci, C. inconspicua, Cladosporium cladosporioides, Debaromyces hansenii, Fomes fomentarius, Galactomyces candidus, Gymnoascus reesii, Chaetomium globosum, Kluyveromyces marxianus, Metarhizium anisopliae, Penicillium aurantiogriseum, P. camemberti, P. freii, P. polonicum, P. viridicatum, Pichia kudriavzevii, Sordaria alcina, Trichosporon lactis and Yarrowia lipolytica.

Development of a scientific study for accessing the criteria under Commission Regulation (EC) 2073/2005 on traditional Slovak sheep cheese "bryndza"

A scientific shelf-life study for Listeria monocytogenes in the typical Slovak cheese "bryndza" was performed in accordance with the requirements of the Commission Regulation (EC) 2073/2005. Based on the previous positive findings of L. monocytogenes in the final products, the producer decided to perform laboratory tests, the results of which would allow him a different evaluation of these positive results. Both the physico-chemical (pH, a<sub>w)</sub> and microbiological examinations of "bryndza" cheese stored at 5.8–6.2°C were performed every two days till the end of the product shelf-life (7 days). Microbiological analyses were performed after artificial contamination of the final product with a mixture of three L. monocytogenes strains. The growth potential of L. monocytogenes was calculated as the difference in the counts of this bacterium between the last day and the first day of the test. The Slovak traditional "bryndza" cheese has been found not to support the growth of L. monocytogenes. Thus, the counts of L. monocytogenes must not exceed 50 CFU/g at the beginning and 20 CFU/g at the end of the product shelf-life in order to ensure its safety for the consumer.

Batch and continuous hydrolysis of ovine whey proteins

The aim of this work was to compare batch and continuous hydrolysis of ovine whey proteins in a membrane reactor and to evaluate influence of thermocalcic precipitation before hydrolysis. Reconstituted dried ovine whey from lump cheese production (a traditional Slovak cheese) was used as a substrate for hydrolysis by Promod 439L protease from Bacillus licheniformis. Initial substrate had 2% proteins and pH 8.5. Hydrolysis was performed directly (i) or after a pre-treatment (ii) comprising thermocalcic precipitation at 50 °C followed by microfiltration at 40 °C, batch-wise or in a membrane ultrafiltration (UF) reactor equipped with 8 kDa ceramic membrane. Mean residence time of 60 min was maintained after batch pre-hydrolysis of the substrate in the first 60 min period, during which the permeate was recirculated. During the subsequent UF, the reaction volume was kept constant by a continuous feeding of the substrate. After the pre-treatment (ii), crude protein decreased by 25%. Degree of hydrolysis was 9.2% (i) and 8.8% (ii) after 60 min at stand-alone batch hydrolysis, and 10.0% (i) and 9.0% (ii) after 60 min at batch-like pre-hydrolysis in the membrane reactor. After 180 min, the degree of hydrolysis (DH) in the UF permeate was 15.2% (i) and 13.0% (ii); the nitrogen content was comparable for (i) and (ii). Without the pre-treatment, (i) required three times higher pressure (0.3 MPa) in comparison with than (ii) to maintain residence time. The batch hydrolysis treatment resulted in 11.9% (i) and 14.6% (ii) DH after 180 min. At least 50% of enzyme can be saved using the membrane reactor in comparison with batch hydrolysis and the product does not contain enzyme and unhydrolyzed proteins.