Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 degrees C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology 180 5947-5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.     

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.     

Cross-inhibition among wild strains of Lactococcus lactis isolated from the same ecological niche.

The cross-inhibition between 23 Lactococcus lactis subsp. lactis strains and 9 L. lactis subsp. cremoris strains with different randomly amplified polymorphic DNA patterns, all isolated from the same ecological niche--cheese made in the spring at a single factory from raw milk without added lactic starter cultures-was investigated. Cross-inhibition, as determined by the agar well diffusion assay, was recorded in 130 cases (12.7%) out of 1.024 total cases, with 109 cases due to supernatants of L. lactis subsp. lactis strains and 21 cases due to supernatants of L. lactis subsp. cremoris strains. L. lactis strains isolated in April, May, and June showed differences in their inhibitory activities, with cross-inhibition against each other in 34.7, 14.1, and 6.1% of the cases, respectively. Polymerase chain reaction techniques using specific primers for nisin, lacticin 481, and lactococcin A only revealed the presence of the structural gene of lacticin 481 in two L. lactis subsp. lactis strains.     

发酵剂对双蛋白干酪理化特性及风味的影响

在牛乳和添加豆乳(质量分数10%)的牛乳中分别使用筛选发酵剂与商品发酵剂进行切达干酪生产,并对成熟干酪的理化成分、质地、风味成分和感官特性进行分析。结果表明,豆乳的添加对干酪的质地、风味和感官特性均无不良影响,而应用筛选发酵剂L. lactis subsp. cremoris QH27-1和L. lactis subsp. lactis XZ3303生产双蛋白切达干酪对干酪的品质有一定的改善作用,可将其应用于双蛋白干酪生产中。     

Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA

Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.     

PCR detection of Bifidobacterium strains and Streptococcus thermophilus in feces of human subjects after oral bacteriotherapy and yogurt consumption

Streptococcus thermophilus, Bifidobacterium infantis Y1 and Bifidobacterium breve Y8 strains were identified and enumerated by PCR assay in human fecal samples after intake of the pharmaceutical preparation VSL-3 or yogurt. ThI/ThII primer set, specific for S. thermophilus, was selected testing its specificity against several strains of enterococci, streptococci and other genera colonizing the human intestine. A culture-independent PCR protocol, developed in this study, allowed to directly detect and enumerate S. thermophilus in human feces, excluding culture-based techniques or time consuming DNA isolation and purification procedures. Intestinal persistence of S. thermophilus was studied in feces of 10 healthy subjects given VSL-3 or yogurt. Streptococcal population was detected after 3 days of administration and persisted for 6 days after the treatment suspension. In the same trial, the colonization kinetics of B. infantis Y1 and B. breve Y8 were studied by amplification of colonies with the strain-specific primer sets InfY-BV.L/R and BreY-BV.R/L, showing a host-dependent transient colonization behaviour. PCR analysis of feces from 10 patients affected by inflammatory bowel diseases (IBD) and treated with VSL-3 for 2 months showed a colonization pattern of S. thermophilus, B. infantis Y1 and B. breve Y8 similar to that observed with the healthy subjects.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

Evidence of a relationship between autolysis of starter bacteria and lipolysis in cheddar cheese during ripening

Cell viability, autolysis and lipolysis were studied in Cheddar cheese made using Lactococcus lactis subsp. cremoris AM2 or Lactococcus lactis subsp. cremoris HP. Cheddar cheese was made in triplicate over a 3 month period and ripened for 238 days at 8 degrees C. Cell viability in cheese was lower for AM2 (a non-bitter strain) than for strain HP (a bitter strain). Autolysis, monitored by the level of the intracellular marker enzyme, lactate dehydrogenase (EC 1.1.1.27) in cheese 'juice' extracted by hydraulic pressure, was much greater in the cheese made using AM2 than that made with HP. Lipolysis was determined by the increase during ripening of individual free fatty acids (FFA) from butyric (C4:0) to linolenic acid (C18:3) measured using a high performance liquid chromatographic technique. Levels of individual FFA from butyric (C4:0) to linolenic (C18:3) acids increased significantly (P<0.05) during ripening in cheeses made with either starter culture. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFA throughout ripening in all cheeses. Levels of caprylic (C8:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids were significantly higher (P<0.05) in cheeses manufactured with Lc. lactis subsp. cremoris AM2 than in cheeses manufactured with Lc. lactis subsp. cremoris HP. Differences in levels of lipolysis between strains was not due to differences in the specific lipolytic or esterolytic activities in cell free extracts of the strains as measured by activity on triolein (lipase) and p-nitrophenylbutyrate (esterase) substrates. Therefore, evidence is provided for a relationship between the extent of starter cell autolysis and the level of lipolysis during Cheddar cheese ripening.     

Impact of autolytic, proteolytic, and nisin-producing adjunct cultures on biochemical and textural properties of cheddar cheese

The effect of incorporating a highly autolytic strain (Lactobacillus delbrueckii subsp. bulgaricus UL12) a proteolytic strain (Lactobacillus casei subsp. casei L2A), or a nisin Z-producing strain (Lactococcus lactis, subsp. lactis biovar diacetylactis UL719) into Cheddar cheese starter culture (Lactococcus lactis KB and Lactococcus cremoris KB) on physicochemical and rheological properties of the resultant cheeses was examined. Cheeses were ripened at 7 degrees C and analyzed over a 6-mo period for viable lactococcal and lactobacilli counts, pH, titratable acidity (TA), lipolysis, proteolysis, and textural characteristics. The combination of the nisin-producing strain and autolytic adjuncts significantly increased the production of water-soluble nitrogen, free amino acids, and free fatty acids. The effect of Lc. diacetylactis UL719 alone or of Lb. casei L2A on water-soluble nitrogen and free amino acid contents were also significant, whereas their effect on free fatty acids was not. Viable counts of Lb. bulgaricus UL12 were significantly reduced in the presence of Lc. diacetylactis UL719. Lactobacilli-containing cheeses showed significantly lower values for hardness, fracturability, and springiness. It could be concluded that the addition of Lb. bulgaricus UL12 together with a nisin-producing strain produces a greater increase in cheese proteolysis and an improvement in Cheddar cheese texture.     

Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production, and expression of pediocin PA-1 in Lactococcus lactis

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.     

Process for improving the quality of direct acidified Cottage cheese

The effects of plain and fermented curd dressing ripened by single (Lactococcus lactis subsp. lactis biovar. diacetylactis) as well as mixed‐strain starter cultures (L. lactis subsp. lactis; L. lactis subsp. cremoris; L. lactis subsp. lactis biovar. diacetylactis:: 1:1:1), different levels of fat (18–24%) in curd dressing and inoculation rate (1–5%) on direct acidified Cottage cheese were observed. Ripened curd dressing containing 22% fat and mixed‐strain starter cultures at 3% imparted a pleasant acidic note, delicate overtones of diacetyl, improved the body and texture, visual appearance and thereby enhanced the overall quality of the product.     

Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture

Hispánico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4:1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0-1.0 g/kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0.11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6 x 10(7) cfu/g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1.0 g bacteriocin-producing culture/kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1.0 g bacteriocin-producing culture/kg were 1.80-fold and 2.17-fold those in control cheese of the same age. Inoculating milk with 1.0 g/kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.     

Comparison of newly isolated strains of Lactobacillus delbrueckii subsp. lactis for hydrogen peroxide production at 5 degrees C

Isolates of Lactobacillus delbrueckii subsp. lactis obtained from raw milk samples were compared for the ability to produce hydrogen peroxide (H2O2) at 5 degrees C. Nineteen out of 101 lactobacilli isolated were identified as L. delbrueckii subsp. lactis. The isolates of L. delbrueckii subsp. lactis from most raw milk samples produced more H2O2 than did isolates of other species of lactobacilli from the same samples. Seven isolates of L. delbrueckii subsp. lactis, which produced the highest levels of H2O2 at 5 degrees C were selected for comparison with a laboratory strain, L. delbrueckii subsp. lactis I. In 24 h, isolate RM2-5 produced 7.0 microg/10(9) cfu in buffer containing 5 mM sodium lactate and 4.4 microg/10(9) cfu in buffer containing 5 mM glucose. Three other isolates also produced more H2O2 on sodium lactate than on glucose. However, three remaining new isolates produced more H2O2 on glucose than on sodium lactate. All seven of the most active new isolates of L. delbrueckii subsp. lactis produced significantly higher concentrations of H2O2 than did L. delbrueckii subsp. lactis I in both solutions. Strain RM2-5 produced more H2O2 than did the other six most active newly isolated strains of L. delbrueckii subsp. lactis in this comparison.     

Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture

Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispánico cheese, a semihard variety. Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added for cheese manufacture. The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments. The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture. Tyramine was detected in all batches of cheese from day 30. Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age. After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches. Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses. The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated. Phenylethylamine and tryptamine were not found in any of the samples.     

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) production in simple media by lactic acid bacterium, Lactococcus lactis subsp. cremoris IFO 3427

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) was detected in simple media prepared with heated sugar and amino acid solution as a result of the Maillard reaction. However, an increase in the amount of HDMF was observed in the media fermented by Lactococcus lactis subsp. cremoris IFO 3427. 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) was not detected in all the simple media examined in this study.     

Effects of administration of Bifidobacterium animalis subsp. lactis GCL2505 on defecation frequency and bifidobacterial microbiota composition in humans

The aim of this study was to evaluate the changes in endogenous bifidobacteria and administered Bifidobacterium animalis subsp. lactis (B. lactis) GCL2505 (GCL2505) in the intestine after administration of GCL2505 by means of a randomized, placebo-controlled double-blind, cross-over study. An increase in the number of total bifidobacteria (the sum of B. bifidum, B. breve, B. longum subsp. longum, B. adolescentis, B. anglatum, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum subsp. infantis and B. lactis) in the feces were observed after administration of GCL2505 using species- and subspecies-specific real-time polymerase chain reaction analysis. However, the number of endogenous bifidobacteria species (excluding B. lactis) remained unchanged. B. lactis also became the predominant bifidobacterial species. Taking into account the number of GCL2505 administered, the findings further suggested that GCL2505 proliferated in the intestine. In addition, the defecation frequency increased during GCL2505 administration compared with the placebo. Moreover, a single administration study (n=17) clearly demonstrated that GCL2505 successfully reached the intestine before proliferating at least 10-fold. This is the first report to show an increase in intestinal bifidobacteria, with no changes to the endogenous species, and improvements in constipation following proliferation of administered bifidobacteria.     

Effect of growth of a commercial starter culture on growth of Staphylococcus aureus and thermonuclease and enterotoxins (C1 and C2) production in broth cultures

Staphylococcus aureus strains FRI 137 (enterotoxin C1 producer) and FRI 361 and L 2 (enterotoxin C2 producers) were grown alone and in the presence of a mixed commercial starter culture (Streptococcus lactis, Streptococcus cremoris and Streptococcus lactis subsp. diacetylactis). Lactic acid bacteria had a slight inhibitory effect on S. aureus population and only during the late stages of growth. In contrast, enterotoxin synthesis was strongly inhibited, inhibition at 18 h being 89% (FRI 137), 80% (FRI 361) and 69% (L 2). Enterotoxin C1 was produced and accumulated during all phases of growth in pure and mixed culture, but associative growth resulted in reduction of enterotoxin C2 after 24-36 h. In mixed culture, high producers of thermonuclease (FRI 137 and L 2) showed an early decrease in enzyme activity followed by an increase, but it never reached levels attained in pure culture. Thermonuclease was detected whenever enterotoxin was detected, but production curves did not parallel each other.     

新型酸奶发酵剂的研制

研究了制备酸奶发酵剂的菌种按不同比例配合所形成的新型发酵剂的特性。通过对专用酸奶菌种StreptococcilsSalirarussubsp.cremoris和Lactobacillusdelbrueekiisubsp.bugaricus的原有研究筛选出的4种组合基础之上,对菌种SE1-2进行活化、扩培、保存并与以上2菌种以不同比例形成新的组合,经过研究筛选出1种最优比例用于酸奶生产,结果效果良好、风味独特。最终认为Lactobacillusdelbrueekiisubsp.bugaricus∶StreptococcilsSalirarussubsp.cremoris∶SE1-2为1∶8∶2是最佳比例。     

Fatty acid membrane composition and activation of glycine-betaine transport in Lactococcus lactis subjected to osmotic stress

Lactococcus lactis subsp. cremoris NCDO763 accumulates glycine-betaine (betaine) when submitted to an osmotic stress with NaCl. Betaine transport activity increases with the extent of the osmotic upshock but also with growth temperature, and supplementation of the medium by Tween-80. Fatty acid analysis of the lipid fraction of L. lactis NCDO763 reveals significant modifications of the fatty acid composition of the membrane when cells are submitted to osmotic stress, high temperature or Tween-80 medium supplementation. The main modification in L. lactis membrane fatty acid composition in response to high osmolality is the increase of Cyclopropane Fatty Acid (CFA) deltaC19:0, whereas Unsaturated/Saturated ratio remains unchanged.     

A washed-curd goat's cheese as a vehicle for delivery of a potential probiotic bacterium: Lactobacillus delbrueckii subsp. lactis UO 004

This study characterizes the probiotic properties of Lactobacillus delbrueckii subsp. lactis UO 004 and examines its suitability for making cheese. This strain was isolated from infant feces and shows interesting features, such as acid and bile tolerance, adherence to intestinal epithelial cells, and inhibition of the growth of certain enteropathogens, that support its potential use as a probiotic strain. In this regard, the suitability of a washed-curd cheese (Vidiago type) made with goat's milk as a delivery system for this probiotic strain was assessed. Lactobacillus delbrueckii subsp. lactis UO 004 was incorporated into a starter culture (IPLA 001). Changes in the overall composition of control and experimental cheeses were determined during ripening through bacteriological, chemical, high-performance liquid chromatography, and gas chromatography analyses. Slight changes in the gross composition and appreciable differences in the flavor compounds profile were observed between control and experimental cheeses. This strain was capable of surviving at high cell numbers (10(8) to 10(9) CFU/g) in cheeses after 28 days of ripening without adversely affecting sensory criteria or appearance of the cheese, thus satisfying the criteria for a probiotic food product.